Inhibitors of nucleoside metabolism

ABSTRACT

The present invention provides compounds having the formula:  
                 
 
     wherein A is CH or N; B is chosen from OH, NH 2 , NHR, H or halogen; D is chosen from OH, NH 2 , NHR, H, halogen or SCH 3 ; R is an optionally substituted alkyl, aralkyl or aryl group; and X and Y are independently selected from H, OH or halogen except that when one of X and Y is hydroxy or halogen, the other is hydrogen; and Z is OH or, when X is hydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ, Q is an optionally substituted alkyl, aralkyl or aryl group; or a tautomer thereof; or a pharmaceutically acceptable salt thereof; or an ester thereof; or a prodrug thereof; and compounds having the formula:  
                 
 
     wherein A, X, Y, Z and R are defined for compounds formula (I) where first shown above; E is chosen from CO 1 H or a corresponding salt form, CO 2 R, CN, CONH 2 , CONHR or CONR 2 ; and G is chosen from NH 2 , NHCOR, NHCONHR or NHCSNHR; or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or an ester thereof, or a prodrug thereof.  
     The present invention also provides the use of the above compounds as pharmaceuticals, pharmaceutical compositions containing the compounds and processes for preparing the compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 08/949,388, filed Oct. 14, 1997, the contents of which arehereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The invention relates to certain nucleoside analogues, the use ofthese compounds as pharmaceuticals, pharmaceutical compositionscontaining the compounds and processes for preparing the compounds.

BACKGROUND OF THE INVENTION

[0003] Purine nucleoside phosphorylase (PNP) catalyses thephosphorolytic cleavage of ribo- and deoxyribonucleosides, for example,those of guanine and hypoxanthine to give the correspondingsugar-1-phosphate and guanine, hypoxanthine, or other purine bases.

[0004] Humans deficient in purine nucleoside phosphorylase (PNP) suffera specific T-cell immunodeficiency due to an accumulation of dGTP andits toxicity to stimulated T lymphocytes. Because of this, inhibitorsagainst PNP are immunosuppressive, and are active against T-cellmalignancies. Clinical trials are now in progress using9-(3-pyridylmethyl)-9-deazaguanine in topical form against psoriasis andin oral form for T-cell lymphoma and immunosuppression (BioCrystPharmaceuticals, Inc). The compound has an IC₅₀ of 35 nM for the enzyme.In animal studies, a 50 mg/kg oral dose is required for activity in acontact sensitivity ear swelling assay in mice. For human doses, thiswould mean approximately 3.5 grams for a 70 kg human. With thisinhibitor, PNP is difficult to inhibit due to the relatively highactivity of the enzyme in blood and mammalian tissues.

[0005] Nucleoside and deoxynucleoside hydrolases catalyse the hydrolysisof nucleosides and deoxynucleosides. These enzymes are not found inmammals but are required for nucleoside salvage in some protozoanparasites. Purine phosphoribosyltransferases (PPRT) catalyze thetransfer of purine bases to 5-phospho-α-D-ribose-1-pyrophosphate to formpurine nucleotide 5′-phosphates. Protozoan and other parasites containPPRT which are involved in essential purine salvage pathways. Malignanttissues also contain PPRT. Some protozoan parasites contain purinenucleoside phosphorylases which also function in purine salvagepathways. Inhibitors of nucleoside hydrolases, purine nucleosidephosphorylases and PPRT can be expected to interfere with the metabolismof parasites and therefore be usefully employed against protozoanparasites. Inhibitors of PNP and PPRT can be expected to interfere withpurine metabolism in malignant tissues and therefore be usefullyemployed against malignant tissues.

[0006] It is an object of the invention to provide pharmaceuticals whichare very effective inhibitors of PNP, PPRT and/or nucleoside hydrolases.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1: FIG. 1 shows purine nucleoside phosphorylase activity withtime at a range of concentrations of the product of Example 1 (CompoundIb).

[0008]FIG. 2: FIG. 2 shows fitting of a purine nucleoside phosphorylaseactivity progress curve to the kinetic model.

[0009]FIG. 3: FIG. 3 shows K_(i)* determination by the curve fit methodfor Compound Ib inhibition of bovine purine nucleoside phosphorylase.

[0010]FIG. 4: FIG. 4 shows a progress curve for bovine purine nucleosidephosphorylase showing slow-onset inhibition by Compound Ib.

[0011]FIG. 5: FIG. 5 shows the effect of oral administration of CompoundIb on the PNP activity of mouse blood.

[0012]FIG. 6: FIG. 6 shows the K_(i) determination for Compound Ib withprotozoan nucleoside hydrolase.

[0013]FIG. 7: FIG. 7 shows the progress curve for purinephosphoribosyltransferase showing slow-onset inhibition by the5′-phosphate of Compound Ib. Inhibition of the malaria enzyme.

[0014]FIG. 8: FIG. 8 shows the K₁* determination for the 5′-phosphate ofCompound Ib inhibition of human purine phosphoribosyltransferase.

DETAILED DESCRIPTION OF THE INVENTION

[0015] In one aspect the invention provides compounds having theformula:

[0016] wherein A is CH or N; B is chosen from OH, NH₂, NHR, H orhalogen; D is chosen from OH, NH₂, NHR, H, halogen or SCH₃; R is anoptionally substituted alkyl, aralkyl or aryl group; and X and Y areindependently selected from H, OH or halogen except that when one of Xand Y is hydroxy or halogen, the other is hydrogen; and Z is OH or, whenX is hydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ, Qis an optionally substituted alkyl, aralkyl or aryl group; or a tautomerthereof; or a pharmaceutically acceptable salt thereof; or an esterthereof; or a prodrug thereof.

[0017] Preferably when either of B and/or D is NHR, then R is C₁-C₄alkyl.

[0018] Preferably when one or more halogens are present they are chosenfrom chlorine and fluorine.

[0019] Preferably when Z is SQ or OQ, Q is C₁-C₅ alkyl or phenyl.

[0020] Preferably D is H, or when D is other than H, B is OH.

[0021] More preferably, B is OH, D is H, OH or NH₂, X is OH or H, Y isH, most preferably with Z as OH, H or methylthio, especially OH.

[0022] It will be appreciated that the representation of a compound offormula (I) wherein B and/or D is a hydroxy group used herein is of theenol-type tautomeric form of a corresponding amide, and this willlargely exist in the amide form. The use of the enol-type tautomericrepresentation is simply to allow fewer structural formulae to representthe compounds of the invention.

[0023] The present invention also provides compounds having the formula:

[0024] wherein A, X, Y, Z and R are defined for compounds of formula (I)where first shown above; E is chosen from CO₂H or a corresponding saltform, CO₂R, CN, CONH₂, CONHR or CONR₂; and G is chosen from NH₂, NHCOR,NHCONHR or NHCSNHR; or a tautomer thereof, or a pharmaceuticallyacceptable salt thereof, or an ester thereof, or a prodrug thereof.

[0025] Preferably E is CONH₂ and G is NH₂.

[0026] More preferably, E is CONH₂, G is NH₂, X is OH or H, Y is H, mostpreferable with Z as OH, H or methylthio, especially OH.

[0027] Particularly preferred are the following compounds:

[0028] 1.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol

[0029] 2.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol

[0030] 3.(1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

[0031] 4.(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

[0032] 5.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol

[0033] 6.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol

[0034] 7.(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

[0035] 8.(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

[0036] 9.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol

[0037] 10.(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

[0038] 11.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

[0039] 12.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol

[0040] 13.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol

[0041] 14.(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

[0042] 15.(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

[0043] 16.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol

[0044] 17.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol

[0045] 18.(1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

[0046] 19.(1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

[0047] 20.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol

[0048] 21.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol

[0049] 22.(1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol

[0050] 23.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol

[0051] 24.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol

[0052] 25.(1S)-1-C-(3-amino-2-carboxamido-4-pyrroly)-1,4-dideoxy-1,4-imino-D-ribitol.

[0053] 26.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate

[0054] 27.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate

[0055] 28.(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitol

[0056] Most preferred are compounds Ib and Ic, their tautomers andpharmaceutically acceptable salts.

[0057] The biological availability of a compound of formula (I) orformula (Ia) can be enhanced by conversion into a pro-drug form. Such apro-drug can have improved lipophilicity relative to the compound offormula (I) or formula (Ia), and this can result in enhanced membranepermeability. One particularly useful form of a pro-drug is an esterderivative. Its utility relies upon the action of one or more of theubiquitous intracellular lipases to catalyse the hydrolysis of theseester group(s), to release the compound of formula (I) and formula (Ia)at or near its site of action.

[0058] In one form of a prodrug, one or more of the hydroxy groups in acompound of formula (I) or formula (Ia) can be O-acylated, to make, forexample a 5-O-butyrate or a 2,3-di-O-butyrate derivative.

[0059] Prodrug forms of 5-phosphate ester derivative of a compounds offormula (I) or formula (Ia) can also be made and may be particularlyuseful, since the anionic nature of the 5-phosphate may limit itsability to cross cellular membranes. Conveniently, such a 5-phosphatederivative can be converted to an uncharged bis(acyloxymethyl) esterderivative. The utility of such a pro-drug relies upon the action of oneor more of the ubiquitous intracellular lipases to catalyse thehydrolysis of these ester group(s), releasing a molecule of formaldehydeand the compound of formula (I) or formula (Ia) at or near its site ofaction.

[0060] Specific examples of the utility of, and general methods formaking, such acyloxymethyl ester pro-drug forms of phosphorylatedcarbohydrate derivatives have been described, e.g. Kang et al.,Nucleosides Nucleotides 17 (1998) 1089; Jiang et al., J. Biol. Chem.,273 (1998) 11017; Li et al., Tetrahedron 53 (1997) 12017; and Kruppa etal., Bioorg. Med. Chem. Lett., 7 (1997) 945.

[0061] According to another aspect of the invention, there is provided apharmaceutical composition comprising a pharmaceutically effectiveamount of a compound of the first aspect of the invention.

[0062] Preferably the pharmaceutical composition comprises a compoundchosen from the preferred compounds of the first aspect of theinvention; more preferably the compound is chosen from the morepreferred compounds of the first aspect. Most preferably the compound isthe compound of formula Ib or Ic.

[0063] In another aspect the invention provides methods for treatment ofdiseases or conditions in which it is desirable to decrease the level ofT lymphocyte activity. The methods comprise administering apharmaceutically effective dose of a compound of the invention to apatient requiring treatment.

[0064] The diseases include T-cell malignancies and autoimmune diseasesincluding arthritis and lupus. This aspect of the invention alsoincludes use of the compounds for immunosuppression for organtransplantation and for inflammatory disorders. The invention includesuse of the compounds for manufacture of medicaments for thesetreatments.

[0065] In another aspect the invention provides a method for treatmentand/or prophylaxis of parasitic infections, particularly those caused byprotozoan parasites. Included among the protozoan parasites are those ofthe genera Giardia, Trichomonas, Leishmania, Trypanosoma, Crithidia,Herpetomonas, Leptomonas, Histomonas, Eimeria, Isopora and Plasmodium.An example of a parasitic infection caused by Plasinodium is malaria.The method can be advantageously applied with any parasite containingone or more nucleoside hydrolases inhibited by the compound of theinvention when administered in an amount providing an effectiveconcentration of the compound at the location of the enzyme.

[0066] In another aspect, the invention provides a method of preparingthe compounds of the first aspect of the invention. The method mayinclude one or more of methods (A)-(Z) and (AA)-(AF).

[0067] Method (A): (4-hydroxypyrrolo[3,2-d]pyrimidines and access to5′-deoxy—, 5′-deoxy-5′-halogeno—, 5′-ether and 5′-thio-analogues)

[0068] reacting a compound of formula (II)

[0069] [wherein Z′ is a hydrogen or halogen atom, a group of formula SQor OQ, or a trialkylsilyloxy, alkyldiarylsilyloxy or optionallysubstituted triarylmethoxy group and Q is an optionally substitutedalkyl, aralkyl or aryl group,] (typically Z′ is atert-butyldimethylsilyloxy, trityloxy or similar group) sequentiallywith N-chlorosuccinimide then a sterically hindered base (such aslithium tetramethylpiperadide) to form an imine, then with the anion ofacetonitrile (typically made by treatment of acetonitrile withn-butyllithium) followed by di-tert-butyl dicarbonate. This generates acompound of formula (III)

[0070] [wherein Z′ is as defined for formula (II) where first shownabove] which is then elaborated following the approach used to prepare9-deazainosine [Lim et al., J. Org. Chem., 48 (1983) 780] in which acompound of formula (III) is condensed with (Me₂N)₂CHOBu^(t) andhydrolyzed under weakly acidic conditions to a compound of formula (IV)

[0071] [wherein Z′ is as defined for formula (II) where first shownabove] which is then sequentially condensed with a simple ester ofglycine (e.g. ethyl glycinate) under mildly basic conditions, cyclizedby reaction with a simple ester of chloroformic acid (e.g. benzylchloroformate or methyl chloroformate) and then deprotected on thepyrrole nitrogen by hydrogenolysis in the presence of a noble metalcatalyst (e.g. Pd/C) in the case of a benzyl group or under mildly basicconditions in the case of a simple alkyl group such as a methyl group,to give a compound of formula (V)

[0072] [wherein Z′ is as defined for formula (II) where first shownabove, and R is an alkyl group] (typically R is a methyl or ethyl group)which is then condensed with formamidine acetate to give a compound offormula (VI)

[0073] [wherein Z′ is as defined for formula (II) where first shownabove] which is then fully deprotected under acidic conditions, e.g. bytreatment with trifluoroacetic acid.

[0074] Methods for the preparation of a compourd of formula (II) whereinZ′ is a tert-butyldimethylsilyloxy group are detailed in Furneaux et al,Tetrahedron 53 (1997) 2915 and references therein.

[0075] A compound of formula (II) [wherein Z′ is a halogen atom], can beprepared from a compound of formula (II) [wherein Z′ is a hydroxygroup], by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation (typically with trifluoroaceticanhydride and a base) to give a compound of formula (VII):

[0076] [wherein R is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group and Z′ is a hydroxygroup] which is then either:

[0077] (i) 5-O-sulfonylated (typically with p-toluenesulfonyl chloride,methanesulfonyl chloride or trifluoromethanesulfonic anhydride and abase) to give a compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is an optionally substituted alkyl- oraryl-sulfonyloxy group], then subjected to a sulfonate displacementreaction with a reagent capable of providing a nucleophilic source ofhalide ion (typically nedium, lithium or a tetraalkylammonium fluoride,chloride, bromide, or iodide); or

[0078] (ii) subjected to a reagent system capable of directly replacinga primary hydroxy group with a halogen atom, for example as in theMitsunobu reaction (e.g. using triphenylphosphine, diethylazodicarboxylate and a nucleophilic source of halide ion as above), byreaction with diethylaminosulfur trifluoride (DAST), or by reaction withmethyltriphenoxyphosphonium iodide in dimethylformamide [see e.g.Stoeckler et al, Cancer Res., 46 (1986) 1774] or by reaction withthionyl chloride or bromide in a polar solvent such ashexamethylphosphoramide [Kitagawa and Ichino, Tetrahedron Lett., (1971)87] to give a compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a halogen atom], which is then selectivelyN-deprotected by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the N-protecting group in use.

[0079] A compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a hydroxy group] can also be prepared froma compound of formula (II) [wherein Z′ is a trialkylsilyloxy,alkyldiarylsilyloxy or optionaily substituted triarylmethoxy group], byN-alkyl- or aralkyl-carboxylation or N-acylation as above, thenselective 5-O-deprotection by acid-catalyzed hydrolysis or alcoholysis,catalytic hydrogenolysis, or treatment with a source of fluoride ion (egtetrabutylammonium fluoride) as required for the 5-O-protecting group inuse.

[0080] The compound of formula (II) [wherein Z′ is a hydrogen atom] canbe prepared from either:

[0081] (i) a 5-hydroxy compound of formula (VII) [wherein R is an alkyl-or aralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a hydroxy group], by formation and radicaldeoxygenation of a 5-O-thioacyl derivative; or

[0082] (ii) a 5-deoxy-5-halogeno-compound of formula (VII) [wherein Z′is a chlorine, bromine or iodine atom] by reduction, either using ahydride reagent such as tributyltin hydride under free radicalconditions, or by catalytic hydrogenolysis, typically with hydrogen overa palladium catalyst; followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use.

[0083] A compound of formula (II) [wherein Z′ is an optionallysubstituted alkylthio, aralkylthio or arylthio group] can be prepared byreaction of a 5-deoxy-5-halogeno or a 5-O-sulfonate derivative offormula (VII) [wherein R is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group and Z′ is a halogenatom or an optionally substituted alkyl- or aryl-sulfonyloxy group]mentioned above, with an alkali metal or tetraalkylammonium salt of thecorresponding optionally substituted alkylthiol, aralkylthiol orarylthiol followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use [see e.g. Montgomery etal., J. Med. Chem., 17 (1974) 1197].

[0084] The compound of formula (II) [wherein Z′ is a group of formulaOQ, and Q is an optionally substituted alkyl, aralkyl or aryl group] canbe prepared from a 5-hydroxy compound of formula (VII) [wherein R is analkyl- or aralkyl-oxycarbonyl group or an optionally substituted alkyl-or aryl-carbonyl group and Z is a hydroxy group], by

[0085] (i) reaction with an alkyl or aralkyl halide in the presence of abase (e.g. methyl iodide and sodium hydride, or benzyl bromide andsodium hydride, in tetrahydrofuran as solvent); or

[0086] (ii) sequential conversion to a 5-O-sulfonate derivative (asabove) and reaction with an alkali metal or tetraalkylammonium salt ofthe desired phenol, followed by selective N-deprotection by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the N-protecting group in use.

[0087] It will be appreciated that the conversions above areconventional reactions employed in carbohydrate chemistry. Manyalternative reagents and reaction conditions can be employed that willeffect these conversions, and references to many of these can be foundin the Specialist Periodical Reports “Carbohydrate Chemistry”, Volumes1-28, published by the Royal Society of Chemistry, particularly in thechapters on Halogeno-sugars, Amino-sugars, Thio-sugars, Esters,Deoxy-sugars, and Nucleosides.

[0088] Method (B): (2-amino-4-hydroxypyrrolo[3,2-d]pyrimidines)

[0089] reacting a compound of formula (V) [wherein Z′ is as defined forformula (II) where first shown above, and R is an alkyl group] withbenzoyl isothiocyanate then methyl iodide in the presence of a base(e.g. DBU or DBN) following the approach used to prepare9-deazaguanosine and its derivatives (see e.g. Montgomery et al., J.Med. Chem., 36 (1993) 55, Lim et al., J. Org. Chem., 48 (1983) 780, andreferences therein] to give a compound of formula (VIII)

[0090] [wherein Z′ is a trialkylsilyloxy, alkyldiarylsilyloxy oroptionally substituted triarylmethoxy group, a hydrogen or halogen atom,SQ or OQ wherein Q is an optionally substituted alkyl, aralkyl or arylgroup and R is an alkyl group] (typically Z′, when a protected hydroxygroup, is a tert-butyldimethylsilyloxy, trityloxy or similar group, andR is a methyl or ethyl group) which is then cyclized in the presence ofammonia to give a separable mixture of compounds of formula (IX)

[0091] [wherein D is an amino or methylthio group, and Z′ and R are asdefined for formula (VIII) where first shown above, or Z′ is a hydroxygroup] (where for example a tert-butyldimethylsilyloxy group has beencleaved under the reaction conditions) and the product of formula (IX)[wherein D is an amino or methylthio group] is fully deprotected underacidic conditions by the procedures set out in Method (A).

[0092] Method (C):(4-aminopyrrolo[3,2-d]pyrimidines)

[0093] reacting a compound of formula (IV) [wherein Z′ is as defined forformula (II) where first shown above] with aminoacetonitrile undermildly basic conditions, cyclization of the product by reaction with asimple ester of chloroformic acid (typically benzyl chloroformate ormethyl chloroformate) to give a compound of formula (X)

[0094] [wherein Z′ is a trialkylsilyloxy, alkyldiarylsilyloxy oroptionally substituted triarylmethoxy group, a hydrogen or halogen atom,SQ or OQ wherein Q is an optionally substituted alkyl, aralkyl or arylgroup and R is an aralkyl or alkyl group] (typically Z′, when aprotected hydroxy group, is a tert-butyldimethylsilyloxy, trityloxy orsimilar group, and R is a benzyl or methyl group) which is thendeprotected on the pyrrole nitrogen by hydrogenolysis in the presence ofa noble metal catalyst (e.g. Pd/C) in the case of a benzyl group orunder mildly basic conditions in the case of a simple alkyl group suchas a methyl group, and processed as described above for thetransformation (V)→(VI)→(I) or (V)→(VIII)→(IX)→(I). This method followsthe approach used to prepare 9-deazaadenosine and its analogues [Lim andKlein, Tetrahedron Lett., 22 (1981) 25, and Xiang et al., NucleosidesNucleotides 15 (1996) 1821].

[0095] Method (D): (7-hydroxypyrazolo[4,3-d]pyrimidines—Daves'methodology)

[0096] reacting a compound of formula (II) [as defined where first shownabove] sequentially with N-chlorosuccinimide and a hindered base (suchas lithium tetramethylpiperidide) to form an imine, then condensing thiswith the anion produced by abstraction of the bromine or iodine atomfrom a compound of formula (XIb) or (XIc)

[0097] [wherein R³ is a bromine or iodine atom and R⁴ is atetrahydropyran-2-yl group) typically using butyllithium or magnesium,to give a product which is then fully deprotected under acidicconditions (as in Method (A)). Methods for preparing compounds offormula (XIb) and (XIc) and mixtures thereof are described in Zhang andDaves, J. Org. Chem., 57 (1992) 4690, Stone et al., J. Org. Chem., 44(1979) 505, and references therein.

[0098] It will be appreciated that while the tetrahydropyran-2-yl groupis favoured as the protecting group for this reaction, otherO,N-protecting groups can be used, and that this method will also beapplicable to the synthesis of analogous pyrazolo[4,3-d]pyrimidinesbearing substituents at position-5 and/or -7 of thepyrazolo[4,3-d]pyrimidine ring independently chosen from a hydroxygroup, an amino, alkylamino, or aralkylamino group or a hydrogen atomusing analogues of compounds of formula (XIb) and (XIc) in which theionizable hydrogen atoms of any hydroxy or amino groups have beenreplaced by a suitable protecting groups.

[0099] Method (E): (7-hydroxypyrazolo[4,3-d]pyrimidines—Yokoyama method)

[0100] subjecting a 5-O-ether protected2,3-O-isopropylidene-D-ribofuranose derivative, where the 5-ethersubstituent is typically a trialkylsilyl, alkyldiarylsilyl, anoptionally substituted triarylmethyl or an optionally substitutedaralkyl group, particularly a tert-butyldimethylsilyl,tert-butyldiphenylsilyl, triisopropylsilyl, trityl or benzyl group, tothe following reaction sequence:

[0101] (i) condensation with the anion produced by abstraction of thebromine or iodine atom from a compound of formula (XIb) or (XIc) fromMethod (D);

[0102] (ii) oxidation of the resulting diol to a diketone, typicallyusing a Swern oxidation or a variant thereof using adimethylsulfoxide-based oxidant (e.g. using a dimethylsulfoxide andtrifluoroacetic anhydride reagent combination in dichloromethanesolution at low temperature, typically −78° C., followed bytriethylamine and warming to room temperature);

[0103] (iii) double reductive amination to form a1,4-dideoxy-1,4-imino-D-ribitol moiety, typically with sodiumcyanoborohydride and ammonium formate, ammonium acetate orbenzhydrylamine in methanol; and

[0104] (iv) removal of the protecting groups by acid-catalyzedhydrolysis (e.g. with 70% aqueous trifluoroacetic acid) and if required(as in the case of the product made with benzhydrylamine or where anoptionally substituted aralkyl group has been used for protecting theprimary hydroxyl group in the iminoribitol moiety) hydrogenolysis over ametal catalyst (typically a palladium catalyst) or if desired (as in thecase of silyl ether protecting group) exposure to a reagent capable ofacting as a source of fluoride ion, e.g. tetrabutylammonium fluoride intetrahydrofuran or ammonium fluoride in methanol). Conditions suitablefor effecting this sequence of reactions are reported in Yokoyama etal., J. Org. Chem., 61 (1996) 6079, and conditions for double reductiveamination with ammonium acetate or benzhydrylamine can be found inFurneaux et al., Tetrahedron 42 (1993) 9605 and references therein.

[0105] Method (F): (7-hydroxypyrazolo[4,3-d]pyrimidines—the Kalvodamethod)

[0106] reacting a compound of formula (II) [as defined where first shownabove] sequentially with N-chlorosuccinimide and a hindered base (suchas lithium tetramethylpiperadide) to form an imine, then with acombination of trimethylsilyl cyanide and a Lewis acid (typically borontrifluoride diethyl etherate) followed by acid catalyzed hydrolysis togive a compound of formula (XII)

[0107] [wherein Z′ is a hydrogen or halogen atom, a hydroxy group, or agroup of formula SQ or OQ where Q is an optionally substituted alkyl,aralkyl or aryl group] which is then converted by sequential selectiveN-protection (typically with trifluoroacetic anhydride, di-tert-butyldicarbonate, benzyl chloroformate, or methyl chloroformate and a base),and O-protection with an acyl chloride or anhydride and a base(typically acetic anhydride or benzoyl chloride in pyridine) to asuitably protected derivative of formula (XIII)

[0108] [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group, Z′ is a hydrogenor a halogen atom, a group of formula SQ or OQ where Q is an optionallysubstituted alkyl, aralkyl or aryl group, or a group of formula R²O, andR² is an alkylcarbonyl or optionally substituted arylcarbonyl group](typically R¹ will be a trifluoroacetyl, tert-butoxycarbonyl orbenzyloxycarbonyl group, and R² will be an acetyl or benzoyl group).

[0109] The carboxylic acid moiety in the resulting compound of formula(XIII) is then transformed into a pyrazolo[4,3-d]pyrimidin-7-one-3-ylmoiety following the method described by Kalvoda [Collect. Czech. Chem.Commun., 43 (1978) 1431], by the following sequence of reactions:

[0110] (i) chlorination of the carboxylic acid moiety to form an acylchloride, typically with thionyl chloride with a catalytic amount ofdimethylformamide in an inert solvent;

[0111] (ii) use of the resulting acyl chloride to acylate hydrogencyanide in the presence of tert-butoxycarbonyltriphenylphosphorane (i.e.Ph₃P═CHCO₂Bu^(t)) to give a 3-cyano-2-propenoate derivative;

[0112] (iii) cycloaddition of this with diazoacetonitrile (which can beprepared from aminoacetonitrile hydrochloride and sodium nitrite) withconcomitant elimination of hydrogen cyanide to give a pyrazolederivative;

[0113] (iv) acid-catalyzed hydrolysis of the tert-butyl ester in thispyrazole derivative to its equivalent carboxylic acid;

[0114] (v) Curtius reaction, typically with phenylphosphoryl azide and2,2,2-trichloroethanol in the presence of triethylamine, which convertsthe carboxylic acid moiety into a 2,2,2-trichloroethoxycarbonylaminogroup (i.e. the product is a carbamate);

[0115] (vi) reductive cleavage of this trichloroethyl carbamate,typically with zinc dust in methanol containing ammonium chloride;

[0116] (vii) condensation of the resulting ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate with formamidine acetateto give a compound of formula (XIV)

[0117] [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group, Z′ is a hydrogenor a halogen atom, SQ or OQ where Q is an optionally substituted alkyl,aralkyl or aryl group, or a group of formula R²O, and R² is analkylcarbonyl or optionally substituted arylcarbonyl group, A is anitrogen atom, B is a hydroxy group and D is a hydrogen atom] which isthen—and O-deprotected by acid- or alkali-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the O- andN-protecting groups in use.

[0118] Method (G): (7-aminopyrazolo[4,3-d]pyrimidines—the Buchananmethod)

[0119] reacting a compound of formula (II) [as defined where first shownabove] sequentially with N-chlorosuccinimide and a hindered base (suchas lithium tetramethylpiperadide) to form an imine, which is thentransformed into a 7-amino-pyrazolo[4,3-d]pyrimidine derivativefollowing the approach used to prepare formycin and its analogues byBuchanan and co-workers [J. Chem. Soc., Perkin Trans. I (1991) 1077 andreferences therein], by the following sequence of reactions:

[0120] (i) addition of 3,3-diethoxyprop-1-ynylmagnesium bromide or3,3-diethoxyprop-1-ynyllithium to the imine;

[0121] (ii) N-protection, typically with trifluoroacetic anhydride,di-tert-butyl dicarbonate, benzyl chloroformate, or methyl chloroformateand a base;

[0122] (iii) mild acid hydrolysis to remove the acid sensitiveO-protecting groups and convert the diethyl acetal moiety into analdehydic moiety;

[0123] (iv) condensation with hydrazine to convert the 3-substitutedprop-2-ynal derivative into a 3-substituted pyrazole derivative;

[0124] (v) acylation, typically with acetic anhydride or benzoylchloride in pyridine;

[0125] (vi) nitration, typically with ammonium nitrate, trifluoroaceticanhydride and trifluoroacetic acid, to produce an 3-substituted1,4-dinitopyrazole derivative;

[0126] (vii) reaction with a reagent capable of delivering cyanide ion,typically sodium cyanide in aqueous ethanol to cause a cine-substitutionof one of the two nitro-groups;

[0127] (viii) reduction of the residual nitro-group, typically withsodium dithionite or by catalytic hydrogenation over a metal catalyst;

[0128] (ix) condensation with formamidine acetate to give a compound offormula (XIV) [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group oran optionally substituted alkyl- or aryl-carbonyl group, Z′ is ahydrogen or a halogen atom, SQ or OQ where Q is an optionallysubstituted alkyl, aralkyl or aryl group, or a group of formula R²Owherein R² is an alkylcarbonyl or optionally substituted arylcarbonylgroup, A is a nitrogen atom, B is an amino group and D is a hydrogenatom] which is then—and O-deprotected by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe O- and N-protecting groups in use.

[0129] Method (H): (2′-deoxy-analogues)

[0130] effecting the overall 2′-deoxygenation of a compound of formula(I) [wherein X and Z are hydroxy groups, Y is a hydrogen atom, and A, Band D are as defined where this formula is first shown above] throughsequential:

[0131] (i) selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation (typically with trilluoroaceticanhydride and a base) of the 1,4-dideoxy-1,4-iminoribitol moiety in sucha compound of formula (I); and

[0132] (ii) 3′,5′-O-protection of the resulting product by reaction with1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane and a base to give acompound of formula (XV):

[0133] [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group, R² is either thesame as R¹ or is a hydrogen atom, and A, B and D are as defined forformula (I) where first shown above]

[0134] (iii) 2′-O-thioacylation of the resulting compound of formula(XV) (typically with phenoxythionocarbonyl chloride and a base; orsodium hydride, carbon disulfide and methyl iodide);

[0135] (iv) Barton radical deoxygenation (typically with tributyltinhydride and a radical initiator);

[0136] (v) cleavage of the silyl protecting group by a reagent capableof acting as a source of fluoride ion, e.g. tetrabutylammonium fluoridein tetrahydrofuran or ammonium fluoride in methanol; and

[0137] (vi) cleavage of the residual N- and O-protecting groups by acid-or alkali-catalyzed hydrolysis or alcoholysis or catalytichydrogenolysis as required for the protecting groups in use.

[0138] Reagents and reaction conditions suitable for conducting the keysteps in this transformation can be found in Robins et al., J. Am. Chem.Soc., 105 (1983) 4059; Solan and Rosowsky, Nucleosides Nucleotides 8(1989) 1369; and Upadhya et al., Nucleic Acid Res., 14(1986) 1747.

[0139] It will be appreciated that a compound of formula (I) has anitrogen atom in its pyrrole or pyrazole ring capable of undergoingalkyl- or aralkyl-oxycarbonylation or acylation during step (i), orthioacylation during step (ii), depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the imino-ribitol moiety, and can be removed during thefinal deprotection step(s). If desired, this approach can be applied toa compound of formula (XV) [as defined above, but additionally bearingN-protecting groups on the pyrazolo- or pyrrolo-pyrimidine moiety].Methods suitable for preparing such N-protected compounds can be foundin Ciszewski et al., Nucleosides Nucleotides 12 (1993) 487; andKambhampati et al., Nucleosides and Nucleotides 5 (1986) 539, as canmethods to effect their 2′-deoxygenation, and conditions suitable forN-deprotection.

[0140] Method (I): (2′-epi-analogues)

[0141] effecting the overall C-2′ epimerization of a compound of formula(I), by oxidizing and then reducing a compound of formula (XV) [asdefined where first shown above] to give compound of formula (XVI):

[0142] [wherein R¹, R², A, B and D are as defined for formula (XV) wherefirst shown above] which may be present in a mixture with the startingalcohol of formula (XV), and then fully deprotecting this compound offormula (XVI) as set out in steps (v) and (vi) of Method (H).

[0143] Reagents and reaction conditions suitable for conducting the keysteps in this transformation can be found in Robins et al., Tetrahedron53 (1997) 447.

[0144] Method (J,: (2′-deoxy-2′-halogeno- and2′-deoxy-2′-epi-2′-halogeno-analogues)

[0145] reacting compound of formula (XV) or (XVI) [as defined wherefirst shown above] by the methods set out in Method (A) for thepreparation of a compound of formula (II) [wherein Z′ is a halogen atom]which involve either:

[0146] (i) 2′-O-sulfonylation and sulfonate displacement with a halideion; or

[0147] (ii) direct replacement of the 2′-hydroxy group with a halogenatom, e.g by the Mitsunobu reaction or reaction with diethylaminosulfurtrifluoride (DAST) to give a compound of inverted stereochemistry atC-2′, which is then fully deprotected as set out in steps (v) and (vi)of Method (H).

[0148] It will be appreciated that a compound of formula (XV) or (XVI)has a nitrogen atom in its pyrrole or pyrazole ring capable ofundergoing sulfonylation during step (i), depending upon the reactionconditions employed. Should such derivatives be formed, the pyrrole orpyrazole N-sulfonate substituents in the resulting derivatives areeither sufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry in the iminoribitol moiety, and can be removedduring the final deprotection step(s).

[0149] If desired, this approach can be applied to a compound of formula(XV) or (XVI) [as defined above, but additionally bearing N-protectinggroups on the pyrazolo- or pyrrolo-pyrimidine moiety). Methods suitablefor preparing such N-protected compounds can be found in Ciszewski etal., Nucleosides Nucleotides 12 (1993) 487; and Kambhampati et al.,Nucleosides and Nucleotides 5 (1986) 539, as can methods to effect2′-O-triflate formation and displacement by halide ion with inversion,and conditions suitable for N-deprotection.

[0150] Method (K): (5′S -deoxy-, 5′-deoxy-5′-halogeno-, 5′-ether and5′-thio-analogues)

[0151] by applying the procedures described in Method (A) for convertinga compound of formula (VII) [wherein R is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group and Z′ is a hydroxy group] into a compound offormula (II) [wherein Z′ is a halogen or hydrogen atom or SQ or OQ whereQ is an optionally substituted alkyl, aralkyl or aryl group alkylthiogroup of one to five carbon atoms] to a compound of formula (XVII):

[0152] [wherein R is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group, Z′ is a hydroxygroup, and A, B and D are as defined for formula (I) where first shownabove] which is then fully deprotected under acidic conditions, e.g. bytreatment with aqueous trifluoroacetic acid.

[0153] Such a compound of formula (XVII) can be prepared from a compoundof formula (I) [wherein X and Z are both hydroxy groups, Y is a hydrogenatom and A, B, and D have the meanings defined for formula (I) wherefirst shown above] in the following two reaction steps, which may beapplied in either order:

[0154] (i) selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation (typically with trifluoroaceticanhydride and a base) of the 1,4-dideoxy-1,4-iminoribitol moiety; and

[0155] (ii) 2′,3′-O-isopropylidenation, which may be effected with avariety of reagents, e.g. acetone and anhydrous copper sulfate with orwithout added sulfuric acid; acetone and sulfuric acid;2,2-dimethoxypropane and an acid catalyst; or 2-methoxypropene and anacid catalyst.

[0156] It will be appreciated that such a compound of formula (I) orformula (XVII) has a nitrogen atom in its pyrrole or pyrazole ringcapable of undergoing sulfonylation, thioacylation, acylation oraralkyl-oxycarbonylation, depending upon the reaction conditionsemployed. Should such derivatives be formed, the pyrrole or pyrazoleN-substituents in the resulting derivatives are either sufficientlylabile that they can be removed by mild acid- or alkali-catalyzedhydrolysis or alcoholysis, or do not interfere with the subsequentchemistry in the iminoribitol moiety, and can be removed during thefinal deprotection step(s).

[0157] Method (L): (2- and 4-aminopyrrolo[3,2-d]pyrimidine and 5- and7-aminopyrazolo[4,3-d]pyrimidine analogues)

[0158] chlorinating a compound of formula (XVIII)

[0159] [wherein R¹ is an alkyl- or aralkyl-oxycarbonyl group or anoptionally substituted alkyl- or aryl-carbonyl group, R² is analkylcarbonyl or optionally substituted arylcarbonyl group, X and Y areindependently chosen from a hydrogen or halogen atom, or a group offormula R²O, except that when one of X or Y is a halogen atom or a groupof formula R²O, the other is a hydrogen atom, Z′ is a group of formulaR²O or, when X is a group of formula R²O, Z′ is a hydrogen or halogenatom, a group of formula R²O or of formula OQ or SQ wherein Q is anoptionally substituted alkyl, aralkyl or an aryl group, A is a nitrogenatom or a methine group, and one of B or D is a hydroxy group, and theother is a chlorine, bromine or hydrogen atom] with a chlorinatingreagent, and then displacing the chlorine atom with a nitrogennucleophile by one of the following methods:

[0160] (i) ammoniolysis, typically using liquid ammonia, concentratedaqueous ammonia, or a solution of ammonia in an alcohol such asmethanol; or

[0161] (ii) conversion first to a triazole derivative, by addition of4-chlorophenyl phosphorodichloridate to a solution of the chloride and1,2,4-triazole in pyridine, and alkaline hydrolysis of both thetetrazole moiety and the ester protecting groups with ammoniumhydroxide;

[0162] (iii) reaction with a source of azide ion, e.g. an alkali metalazide or tetraalkylammonium azide, and reduction of the resultingproduct, typically by catalytic hydrogenation; or

[0163] (iv) reaction with an alkylamine or aralkylamine, such asmethylamine or benzylamine in methanol.

[0164] These conditions are sufficiently basic that O-ester groups willgenerally be cleaved but any residual O- or N-protecting groups can thenbe removed by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the protecting groups in use.

[0165] Suitable chlorinating agents are thionylchloride—dimethylformamide complex [Ikehara and Uno, Chem. Pharm. Bull.,13 (1965) 221], triphenylphosphine in carbon tetrachloride anddichloromethane with or without added 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) ]De Napoli et al., J. Chem. Soc., Perkin Trans.1 (1995) 15 andreferences therein;, phosphoryl chloride (Imai, Chem. Pharm. Bull., 12(1964) 1030], or phenylphosphoryl chloride and sodium hydride.

[0166] Suitable conditions for such an ammoniolysis or a reaction withan alkylamine can be found in Ikehara and Uno, Chem. Pharm. Bull., 13(1965) 221; Robins and Tripp, Biochemistry 12 (1973) 2179; Marumoto etal., Chem. Pharm. Bull., 23 (1975) 759; and Hutchinson et al., J. Med.Chem., 33 (1990) 1919].

[0167] Suitable conditions for conversion of a such a chloride to anamine via a tetrazole derivative can be found in Lin et al., Tetrahedron51 (1995) 1055.

[0168] Suitable conditions for reaction with azide ion followed byreduction can be found in Marumoto et al., Chem. Pharm. Bull., 23 (1975)759.

[0169] Such a compound of formula (XVIII) can be prepared from acompound of formula (I) by selective N-alky- or aralkyl-oxycarbonylation(typically with di-tert-butyl dicarbonate, benzyl chloroformate, ormethyl chloroformate and a base) or N-acylation of the1,4-dideoxy-1,4-iminoribitol moiety and then O-acylation (typically withacetic anhydride or benzoyl chloride in pyridine). It will beappreciated that such a compound of formula (I) has a nitrogen atom inits pyrrole or pyrazole ring capable of undergoing alkyl- oraralkyl-oxycarbonylation or acylation depending upon the reactionconditions employed. Should such derivatives be formed, the pyrrole orpyrazole N-substituents in the resulting derivatives are eithersufficiently labile that they can be removed by mild acid- oralkali-catalyzed hydrolysis or alcoholysis, or do not interfere with thesubsequent chemistry, and can be removed during the final deprotectionstep(s).

[0170] The above chlorination—amination—deprotection sequence can alsobe applied to a compound of formula (XVII) [wherein B is a hydroxygroup, D is a hydrogen atom, Z′ is a hydrogen or halogen atom, or agroup of formula R²O, R² is a trialkylsilyloxy or alkyldibrylsilyloxygroup, or an optionally substituted triarylmethoxy, alkylcarbonyl orarylcarbonyl group, R and A are as defined for formula (XVII) wherefirst shown above]. Suitable conditions for conducting this reactionsequence can be found in Ikehara et al., Chem. Pharm. Bull., 12 (1964)267.

[0171] Method (M): (2,4-dihydroxypyrrolo[3,2-d]pyrimidine and5,7-dihydroxypyrazolo[4,3-d]pyrimidine analogues)

[0172] oxidation of either:

[0173] (i) a compound of formula (XVIII) (wherein R² is a hydrogen atom;X and Y are independently chosen from a hydrogen or halogen atom, or ahydroxy group, except that when one of X or Y is a halogen atom or ahydroxy group, the other is a hydrogen atom; Z′ is a hydroxy group or,when X is a hydroxy group, Z′ is a hydrogen or halogen atom, a hydroxygroup, or OQ; Q is an optionally substituted alkyl, aralkyl or arylgroup; B is a hydroxy group or an amino group; D is a hydrogen atom; andR¹ and A are as defined for formula (XVIII) where first shown above]with bromine in water; or

[0174] (ii) a compound of formula (XVIII) [wherein Z′ is a hydrogen or ahalogen atom, or a group of formula R²O, or OQ; Q is an optionallysubstituted alkyl, aralkyl or aryl group; B is a hydroxy group or anamino group, D is a hydrogen atom and R¹, R², X, Y and A are as definedfor formula (XVIII) where first shown above], with bromine or potassiumpermanganate in water or in an aqueous solvent mixture containing aninert, water-miscible solvent to improve the solubility of thesubstrate, to give a related compound of formula (XVIII) [but wherein Band D are now hydroxy groups], and then removal of any O- andN-protecting groups by acid- or alkali-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the protectinggroups in use.

[0175] Such a compound of formula (XVIII) required for step (i) abovecan be prepared from a compound of formula (I) [wherein Z is Z′, and X,Y, Z, A, B and D are as defined for the required compound of formula(XVIII)] by selective N-alkyl- or aralkyl-oxycarbonylation (typicallywith di-tert-butyl dicarbonate, benzyl chloroformate, or methylchloroformate and a base) or N-acylation (typically with trifluoroaceticanhydride and a base) of the 1,4-dideoxy-1,4-iminoribitol moiety. Thiscan then be converted to the corresponding compound of formula (XVIII)required for step (ii) above by O-acylation (typically with aceticanhydride or benzoyl chloride in pyridine). It will be appreciated thatsuch a compound of formula (I) has a nitrogen atom in its pyrrole orpyrazole ring capable of undergoing alkyl- or aralkyl-oxycarbonylationor acylation depending upon the reaction conditions employed. Shouldsuch derivatives be formed, the pyrrole or pyrazole N-substituents inthe resulting derivatives are either sufficiently labile that they canbe removed by mild acid- or alkali-catalyzed hydrolysis or alcoholysis,or do not interfere with the subsequent chemistry, and can be removedduring the final deprotection step(s).

[0176] Method (N): (4-amino-2-chloropyrrolo[3,2-d]pyrimidine and7-amino-5-chloropyrazolo[4,3-d]pyrimidine analogues)

[0177] chlorinating a compound of formula (XVIII) [wherein B and D arehydroxy groups and R¹, R², X, Y, Z′ and A are as defined for formula(XVIII) where first shown above] to give a correspondingdichloro-derivative of formula (XVIII) [wherein B and D are chlorineatoms], typically with neat phosphorous oxychloride, and then displacingthe more reactive chloro-substituent selectively by ammoniolysis,typically using anhydrous liquid ammonia in a pressure bomb ormethanolic ammonia, which simultaneously cleaves the O-ester protectinggroups. The residual N-protecting group is then removed byacid-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysis asrequired for the protecting groups in use, to give a compound of formula(I) [wherein B is an amino-group and D is a chlorine atom].

[0178] The above dichloro-derivative of formula (XVIII) can be convertedinto a compound of formula (I) [wherein B and D are chlorine atoms] byremoval of the O- and N-protecting groups by acid- or alkali-catalyzedhydrolysis or alcoholysis as required for the protecting groups in use.It will be appreciated that one of the chlorine atoms in theaforementioned compound of formula (XVII) or of formula (I) is quitereactive and that conditions chosen for deprotection must be mild enoughthat they limit unwanted reactions involving this atom.

[0179] Suitable reaction conditions for the key steps in this method canbe found in Upadhya et al., Nucleic Acid Res., 14 (1986) 1747 andKitagawa et al., J. Med. Chem., 16 (1973) 1381.

[0180] Method (O): (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues fromdichloro-compounds)

[0181] hydrolysis of a compound of formula (XVIII) [wherein B and D arechlorine atoms] available as an intermediate from the first reaction ofMethod (N), typically with aqueous potassium hydroxide or sodiumcarbonate, in the presence of an inert, water-miscible solvent such asdioxane to enhance solubility as required, followed by removal of theresidual N-protecting group by acid-catalyzed hydrolysis or alcoholysisor catalytic hydrogenolysis as required for the protecting groups inuse, to give a compound of formula (I) [wherein B is a hydroxy group andD is a chlorine atom].

[0182] Method (P): (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues fromaminochloro-compounds)

[0183] deamination of a compound of formula (XVIII) [wherein B is anamino group, D is a chlorine atom, R¹ is an alkyl- oraralkyl-oxycarbonyl group or an optionally substituted alkyl- oraryl-carbonyl group, R² is a hydrogen atom, Z′═Z and X, Y, Z and A areas defined for formula (I) where first shown above], available as anintermediate following the chlorination and ammonyolysis reactions ofMethod (N), by reaction with nitrosyl chloride, followed by removal ofthe protecting groups as set out in Method (N). Typical reactionconditions can be found in Sanghvi et al., Nucleosides Nucleotides 10(1991) 1417.

[0184] Method (Q): (4-halogenopyrrolo[3,2-d]pyrimidine and7-halogenopyrazolo[4,3-d]pyrimidine analogues)

[0185] reacting a compound of formula (XVIII) [wherein R¹ istert-butoxycarbonyl group, B is a hydroxy group, D is a hydrogen atomand R², X, Y, Z′ and A are as defined for formula (XVIII) where firstshown above] by a method used to prepare halogeno-formycin analogues(Watanabe et al., J. Antibiotic, Ser. A 19 (1966) 93] which involvessequential treatment with:

[0186] (i) phosphorous pentasulfide by heating in pyridine and waterunder reflux to give a mercapto-derivative;

[0187] (ii) methyl iodide to give a methylthio-derivative;

[0188] (iii) a base in a simple alcohol or an aqueous solution of asimple alcohol, e.g. sodium methoxide in methanol, to remove theO-protecting groups; and

[0189] (iv) chlorine, bromine or iodine in absolute methanol to give ahalogeno-derivative which is then N-deprotected by reaction with aqueousacid, typically a concentrated trifluoroacetic acid solution.

[0190] Method (R): (pyrrolo[3,2-d]pyrimidine andpyrazolo[4,3-d]pyrimidine analogues)

[0191] hydrogenolytic cleavage of the chloride intermediate resultingfrom the chlorination reaction used as the first reaction in Method (L),or the chloride intermediate resulting from the chlorination reactionstep (iv) in Method (Q), or the compound of formula (I) produced byMethod (Q), typically using hydrogen over palladium on charcoal as thecatalyst, optionally with magnesium oxide present to neutralize releasedacid, followed by cleavage of any residual O- or N-protecting groups byacid- or alkali-catalyzed hydrolysis or alcoholysis as required for theprotecting groups in use.

[0192] Method (S): (N-alkylated 4-aminopyrrolo[3,2-d]pyrimidine and7-aminopyrazolo[4,3-d]pyrimidine analogues)

[0193] heating an O-deprotected methylthio-derivative produced by step(iii) of Method (Q) with an amine, e.g. methylamine, in absolutemethanol in a sealed tube or bomb, and then removing the N-protectinggroup by reaction with aqueous acid, typically a concentratedtrifluoroacetic acid solution. This method has been used to prepareN-alkylated-formycin analogues [Watanabe et al., J. Antibiotic, Ser. A19 (1966) 93]; or reacting a compound of formula (I) [wherein either Bor D is an amino group] with 1,2-bis[(dimethylamino)methylene]hydrazineand trimethylsilyl chloride in toluene to convert the amino group into a1,3,4-triazole group, hydrolysis to cleave the O-silyl groups (e.g. withacetic acid in aqueous acetonitrile), and displacement of the1,3,4-triazole group with an alkylamine in a polar solvent (e.g. wateror aqueous pyridine). This method has been used to prepareN,N-dimethyl-formycin A [Miles et al., J. Am. Chem. Soc., 117 (1995)5951]; or subjecting a compound of formula (I) (wherein either B or D isan amino group] to an exchange reaction by heating it with an excess ofan alkylamine. This method has been used to prepare N-alkyl-formycin Aderivatives [Hecht et al., J. Biol. Chem., 250 (1975) 7343].

[0194] Method T: (2-chloro-4-hydroxypyrrolo[3,2-d]pyrimidine and5-chloro-7-hydroxypyrazolo[4,3-d]pyrimidine analogues)

[0195] Selective chlorination of dihydroxy compound of formula (XVIII)[wherein B and D are hydroxy groups, and R¹, R², X, Y, Z′ and A are asdefined for formula (XVIII) where first shown above], taking advantageof the greater reactivity of the 4-hydroxy group on a2,4-dihydroxypyrrolo[3,2-d]pyrimidine derivative and the 7-hydroxy groupon a 5,7-dihydroxypyrazolo[4,3-d]pyrimidine derivative, followed byremoval of protecting groups, using the methods set out in Method (N).

[0196] Method U: (2-halogeno-, 4-halogeno- and2,4-dihalogeno-pyrrolo[3,2-d]pyrimidine and 5-halogeno-, 7-halogeno-,and 5,7-dihalogeno-pyrazolo [4,3-d]pyrimidine analogues) diazotizationof a compound of formula (XVIII) [wherein one of B or D is an aminogroup, and the other is independently chosen from an amino group, or ahalogeno or hydrogen atom, and R¹ , R² , X, Y, Z′ and A are as definedfor formula (XVIII) where first shown above] and subsequent reactionusing one of the following procedures:

[0197] (i) with nitrous acid (made in situ from sodium nitrite) in thepresence of a source of halide ion. For replacement of an amino-groupwith a fluorine atom, a concentrated aqueous solution of fluoroboricacid [Gerster and Robins, J. Org. Chem., 31 (1966) 3258; Montgomery andHewson, J. Org. Chem., 33 (1968) 432] or hydrogen fluoride and pyridineat low temperature (e.g. −25 to −30° C.) [Secrist et al., J. Med. Chem.,29 (1986) 2069] can serve both as the mineral acid and the fluoride ionsource; or

[0198] (ii) with an alkyl nitrite, typically tert-butyl or n-butylnitrite, in a non-aqueous solvent in the presence of a source of halideion. For replacement of an amino-group with a chlorine atom, acombination of chlorine and cuprous chloride, or antimony trichloridecan be used in chloroform as solvent [Niiya et al, J. Med. Chem., 35(1992) 4557 and references therein]; or

[0199] (iii) with an alkyl nitrite, typically tert-butyl or n-butylnitrite, in a non-aqueous solvent coupled with photohalogenation. Forreplacement of an amino group with a chlorine, bromine or iodine atom,carbon tetrachloride, bromoform, or diiodomethane have been used asreagent and solvent and an incandescent light source (e.g. a 200 W bulb)has been used to effect photohalogenation [Ford et al., J. Med. Chem.,38 (1995) 1189; Driscoll et al., J. Med. Chem., 39 (1996) 1619; andreferences therein]; to give a corresponding compound of formula (XVIII)[wherein B is a halogen atom a is either a halogen atom or an aminogroup], followed by removal of the protecting groups as set out inMethod (N).

[0200] The same transformations can be effected for a correspondingstarting compound of formula (XVIII) [wherein one of B or D is an aminogroup, and the other is a hydroxy group] if the hydroxy group is firstconverted to a thiol group [Gerster and Robins, J. Org. Chem., 31 (1966)3258]. This conversion can be effected by reaction with phosphorouspentasulfide by heating in pyridine and water under reflux (see Method(Q)).

[0201] Method (V): (4-iodo-pyrazolo[3,2-d]pyrimidine and7-iodopyrazolo[4,3-d]pyrimidine analogues)

[0202] treatment of corresponding chloro-analogue of formula (I)[wherein B is a chlorine atom] with concentrated aqueous hydroiodicacid, following the method of Gerster et al., J. Org. Chem., 28 (1963)945.

[0203] Method (W): (5′-deoxy-5′-halogeno- and 5′-thio-analogues)

[0204] by reacting a compound of formula (XVIII) [wherein R² is ahydrogen atom; X and Y are independently chosen from a hydrogen orhalogen atom, or a hydroxy group, except that when one of X or Y is ahalogen atom or a hydroxy group, the other is a hydrogen atom; Z is ahydroxy group; and R¹, A, B and D are as defined for formula (XVIII)where first shown above] with either

[0205] (i) a trisubstituted phosphine and a disulfide, e.g.tributylphosphine and diphenyl disulfide; or

[0206] (ii) a trisubstituted phosphine (e.g. triphenylphosphine) andcarbon tetrabromide; or

[0207] (iii) thionyl chloride or bromide.

[0208] and then removal of the N-protecting group by acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the protecting group in use.

[0209] Conditions suitable for conducting such selective replacements ofa 5′-hydroxy group with a thio group or a halogen atom can be found inChern et al., J. Med. Chem., 36 (1993) 1024; and Chu et al., NucleosideNucleotides 5 (1986) 185.

[0210] Method (X): (5′-phospho-pyrazolo[3,2-d]pyrimidine and5′-phospho-pyrazolo[4,3-d]pyrimidine analogues)

[0211] reacting a compound of formula (XVII) [wherein R, Z′, A, B and Dare as defined where first shown) with

[0212] (i) a phosphitylation agent, such asN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine, thenoxidizing the phosphite ester to a phosphate ester, e.g. with3-chloroperbenzoic acid; or

[0213] (ii) a phosphorylatiing agent, such as phosphoryl chloride ordibenzylchlorophosphate; and removing the protecting groups, e.g. byhydrogenolysis and treatment under acidic conditions as set out inMethod (A)

[0214] Method (Y): (3-aminopyrrole-2-carboxylic acid and4-amino-1H-pyrazole-5-carboxylic acid analogues)

[0215] fully deprotecting a compound of formula (V) as defined wherefirst shown, or an intermediate ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by step (vi) inMethod (F), by acid- or alkali-catalyzed hydrolysis or alcoholysis orcatalytic hydrogenolysis as required for the O- and N-protecting groupsin use.

[0216] Method (Z): (3-amino-2-cyanopyrroles and4-amino-5-cyano-1H-pyrazoles)

[0217] fully deprotecting a compound of formula (X) as defined wherefirst shown above, or a 4-amino-5-cyanopyrazole intermediate produced bystep (viii) in Method (G), by acid-or alkali-catalyzed hydrolysis oralcoholysis or catalytic hydrogenolysis as required for the O- andN-protecting groups in use.

[0218] Method (AA): (3-aminopyrrole-2-carboxamide and4-amino-1H-pyrazole-5-carboxamide analogues)

[0219] conversion of the cyano-group of a compound of formula (X) asdefined where first shown above, or a 4-amino-5-cyano-1H-pyrazolesintermediate produced by step (viii) in Method (G), into acarboxamido-group, conveniently by reaction with hydrogen peroxide andpotassium carbonate in dimethylsulfoxide, and then fully deprotectingthe resulting product by acid- or alkali-catalyzed hydrolysis oralcoholysis or catalytic hydroenolysis as required for the O- andN-protecting group in use.

[0220] Method (AB): (3-(thio)carbamoylpyrroles and4-5-thio)carbamoyl-1H-pyrazoles)

[0221] reaction of a compound of formula (V) or formula (X) as definedwhere first shown above, or a protected carboxamido-intermediate asprepared in Method (AA), or an intermediate ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by step (vi) inMethod (F), with an isocyanate or isothiocyanate of formula RNCO orRNCS, where R is as defined for compounds of formula (I) and then fullydeprotecting the resulting product by acid- or alkali-catalyzedhydrolysis or alcoholysis or catalytic hydrogenolysis as required forthe O- and N-protecting groups in use.

[0222] Method (AC): (esters of 3-aminopyrrole-2-carboxylic acid and4-amino-1H-pyrazole-5-carboxylic acid analogues)

[0223] converting the carboxylic acid group of a compound of formula(Ia) wherein E is CO₂H into an ester, which can be accomplished by anumber of well known methods for esterification. Conveniently an estercan be made by reaction of the carboxylic acid in acidic solution of thealcohol, e.g., ethanolic hydrogen chloride.

[0224] Method (AD): (3-acylaminopyrroles and 4-acylamino-1H-pyrazoles)

[0225] reaction of a compound of formula (V) or (X) as defined wherefirst shown above, or an intermediate ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by step (vi) inMethod (F), with an acylating agent, e.g. an acyl chloride such asbenzoyl chloride, acid anhydride such as acetic anhydride in thepresence of a base, such as triethylamine, potassium carbonate orpyridine, and then fully deprotecting the resulting product acid- oralkali-catalyzed hydrolysis or alcoholysis or catalytic hydrogenolysisas required for the O- and N-protecting groups in use.

[0226] Method (AE): (N-mono- and N,N-di-substituted3-amino-pyrrole-2-carboxamide and 4-amino-1H-pyrazole-5-carboxamideanalogues)

[0227] converting the carboxylic acid group of a compound of formula(Ia) wherein E is CO₂H into an amide. Conveniently an amide can be madeby carbodiimide induced condensation (e.g. withN,N-dicylcohexylcarbodiimide) of the carboxylic acid with a primary orsecondary amine.

[0228] Method (AF): (N-mono- and N,N-di-substituted3-amino-pyrrole-2-carboxamide and 4-amino-1H-pyrazole-5-carboxamideanalogues)

[0229] condensing a compound of formula (V) as defined where firstshown, or an intermediate ethyl4-amino-3-substituted-1H-pyrazole-5-carboxylate produced by step (vi) inMethod (F), with a primary or secondary amine and fully deprotecting theresulting product by acid- or alkali-catalyzed hydrolysis or alcoholysisor catalytic hydrogenolysis as required for the O- and N-protectinggroups in use.

[0230] It will be appreciated that the approaches outlined in Methods(H), (I), (J), (K) and (W) are equally applicable to the synthesis ofcompounds of formula (Ia) to give analogous variations in the1,4-imino-pentitol moiety.

[0231] Method (AG): (Acyloxymethyl ester prodrugs)

[0232] reacting a 5-phosphate ester of a compound of formula (I) orformula (Ia) with benzylchloroformate in the presence of a base,conveniently aqueous sodium bicarbonate, to form an N-benzyloxycarbonylderivative, reacting this derivative with an acyloxymethyl halide offormula RCO₂CH₂X where R is an alkyl group such as methyl, ethyl, propylor tert-butyl and X is chloride, bromide or iodide, in the presence of abase, to form the 5-phosphate bis(acyloxymethyl) ester. Suitableconditions for the formation of the acetoxymethyl esters, usingacetoxymethyl bromide and diisopropylethylamine in dimethylformamide,can be found in Kruppa et al, Bioorg. Med. Chem. Lett., 7 (1997) 945.

[0233] When desired, e.g. as when the aforementioned N-benzyloxycarbonylderivative is not sufficiently soluble in the reaction solvent, thisderivative may first be converted into the corresponding stannylintermediates, e.g. the bis(tributylstannyl) phosphate derivative byreaction with tributyltin methoxide in methanol, prior to reaction withthe acyloxymethyl halide in the presence of tetrabutylammonium bromide,following the method described by Kang et al., Nucleosides Nucleotides17 (1998) 1089.

[0234] It will be appreciated that the conversion of such a 5-phosphategroup to the corresponding bis(acyloxymethyl) ester can be accomplishedby utilizing O- and or N-protected derivatives of compounds of formula(I) or formula (Ia) if desired, so long as the protecting groups cansubsequently be removed without the use of strongly acidic or stronglybasic conditions. Typically this requires the use of hydrogenolysisconditions for deprotection, so that O- and N-benzyl, —benzyloxymethylor —benzyloxycarbonyl groups are favoured.

[0235] Further Methods

[0236] Compounds of the invention may also be prepared by other methodsas will be apparent to those skilled in the art.

[0237] Further Aspects

[0238] The compounds of the invention are useful both in free base formand in the form of salts. The term “pharmaceutically acceptable salts”is intended to apply to non-toxic salts derived from inorganic ororganic acids including for example salts derived from the followingacids —hydrochloric, sulfuric, phosphoric, acetic, lactic, fumaric,succinic, tartaric, gluconic, citric, methanesulphonic andp-toluenesulphonic acids.

[0239] The compounds of the invention are potent inhibitors of purinenucleoside phosphorylases, nucleoside hydrolases and/orphosphoribosyltransferases. For example, the IC₅₀ values for thecompounds of formula (Ib) and formula (Ic) are less than 0.1 nM for bothcalf spleen PNP and human red blood cell PNP. The examples below providefurther detail of the effectiveness of this inhibitor. Purine nucleosidephosphorylase inhibitory activity can be determined by the coupledxanthine oxidase method using inosine as the purine substrate (H. M.Kalckar, J.) Biol. Chem. 167 (1947) 429-443. Purinephosphoribosyltransferase activity is detected in the same assay usinginosine 5′-phosphate as the substrate. Slow onset inhibitor binding canbe determined using methods such as those described by Merkler et al.,Biochemistry 29 (1990) 8358-64. Parasite nucleoside hydrolase activitymay be measured inter alia by methods disclosed in published PCTinternational patent application W097/31008 and the references citedtherein.

[0240] The potency of the inhibitors of the invention provides importantadvantages over the prior art because of the relatively high activity ofPNP in blood and mammalian tissue. As mentioned above the requireddosage of 9-(3-pyridylmethyl)-9-deazaguanine may be of the order of 3.5grams per dose for a human adult. The present invention provides theadvantage that considerably lower quantities of the compounds arerequired. This allows cost saving and may also reduce unwanted sideeffects.

[0241] The amount of active ingredient to be administered can varywidely according to the nature of the patients and the nature and extentof the disorder being treated. Typically the dosage for an adult humanwill be in the range less than 1 to 1000 milligrams, preferably 0.1 to100 milligrams. The active compound can be administered with aconventional pharmaceutical carrier and may be administered orally, byinjection or topically.

[0242] The preferred route of administration is oral administration. Foradministration by this route the compounds can be formulated into solidor liquid preparations, eg tablets, capsules, powders, solutions,suspensions and dispersions. Such preparations are well known in the artas are other oral dosage forms not listed here. In a preferredembodiment the compounds of the invention are tableted with conventionaltablet bases such as lactose, sucrose and corn starch together with abinder, a disintegration agent and a lubricant. These exipients are wellknown in the art. The binder may be for example corn starch or gelatin,the disintegrating agent may be potato starch or alginic acid and thelubricant may be magnesium stearate. Other components such as colouringagents and flavouring agents may be included.

[0243] Liquid forms for use in the invention include carriers such aswater and ethanol, with or without other agents such as apharmaceutically acceptable surfactant or suspending agent.

[0244] The compounds of the invention may also be administered byinjection in a physiologically acceptable diluent such as water orsaline. The diluent may comprise one or more of other ingredients suchas ethanol, propylene glycol, an oil or a pharmaceutically acceptablysurfactant.

[0245] Compounds of the invention may be applied to skin or mucousmembranes. They may be present as ingredients in creams, preferablyincluding a pharmaceutically acceptable solvent to assist passagethrough the skin or mucous membranes. Suitable cream bases are wellknown to those skilled in the art.

[0246] The compounds of the invention may be administered by means ofsustained release systems for example they may be incorporated into aslowly dissolving tablet or capsule containing a solid or porous ormatrix form from a natural or synthetic polymer.

EXAMPLES

[0247] The following examples further illustrate practice of theinvention. Ratios of solvents are by volume.

Example 1 Preparation of(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol Example 1.1.

[0248] A solution of5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(Furneaux et al, Tetrahedron 53 (1997) 2915 and references therein) (2.0g) in pentane (40 ml) was stirred with N-chlorosuccinimide (1.2 g) for 1h. The solids and solvent were removed and the residue was dissolved indry tetrahydrofuran (40 ml) and cooled to −78° C. A solution of lithiumtetramethylpiperidide (25 ml, 0.4 M in tetrahydrofuran) was added slowlydropwise. The resulting solution was then added via cannula to asolution of lithiated acetonitrile [prepared by the dropwise addition ofaceronitrile (2.08 ml, 40 mmol) to a solution of butyl lithium (29.8 ml,41.8 mmol) in dry tetrahydrofuran (50 ml) at −78° C., followed bystirring for 45 min and then addition of tetramethylpiperidine (0.67 ml,4 mmol)] at −78° C. The reaction mixture was stirred for 15 min thenquenched with water and partitioned between water and chloroform. Theorganic phase was dried and concentrated, and then chromatographyafforded(1S)-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1) (0.83 g).

Example 1.2

[0249] A solution of the product from Example 1.1 (0.80 g) indichloromethane (20 ml) containing di-tert-butyldicarbonate (0.59 g) wasstirred at room temperature for 16 h. The solution was concentrated andthen chromatography afforded(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(2) (0.89 g).

Example 1.3

[0250] To a solution of the product from Example 1.2 (0.88 g) inN,N-dimethylformamide (5 ml) was added tert-butoxybis(dimethylamine)methane (1.5 ml) and the solution was heated at 65-70°C. for 1 h. Toluene (20 ml) was added and the solution was washed (×3)with water, dried and concentrated to dryness. The residue was dissolvedin tetrahydrofuran/acetic acid/water (1:1:1 v/v/v, 40 ml) at roomtemperature. After 1.5 h chloroform (50 ml) was added and the mixturewas washed with water (×2), aqueous sodium bicarbonate, and then driedand evaporated to dryness Chromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-(1-cyano-2-hydroxyethenyl)-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol (3) (0.68 g).

Example 1.4

[0251] Glycine hydrochloride ethyl ester (0.76 g) and sodium acetate(0.9 g) were added to a stirred solution of the product from Example 1.3(0.51 g) in methanol (10 ml). The mixture was stirred at roomtemperature for 16 h and then concentrated to dryness. Chromatography ofthe residue gave the(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-(1-cyano-2-(ethoxycarbonylmethylamino)ethenyl]-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(4) (0.48) g as a diastereomeric mixture.

Example 1.5

[0252] A solution of the product from Example 1.4 (0.28 g) in drydichloromethane (12 ml) containing 1,8-diazabicyclo[5.4.0undec-7-ene(1.5 ml) and benzyl chloroformate (0.74 ml) was heated under reflux for8 h, then cooled and washed with dilute aqueous HCl, aqueous sodiumbicarbonate, dried and concentrated. Chromatography of the residueafforded(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(5) (0.22 g)

Example 1.6

[0253] A solution of the product from Example 1.5 (0.22 g) in ethanol(10 ml) was stirred with 10% Pd/C (50 mg) in an atmosphere of hydrogenfor 3 h. The solids and solvent were removed and the residue wasdissolved in ethanol (10 ml) containing formamidine acetate (0.40 g) andthe solution was heated under reflux for 8 h. The solvent was removedand chromatography of the residue gave(1S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1-C-[4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-1,4-imino-2,3-O-isopropylidene-D-ribitol(6) (156 mg).

Example 1.7

[0254] A solution of the product from Example 1.6 (66 mg) intrifluoroacetic acid (3 ml) was allowed to stand at room temperatureovernight. The solution was concentrated and a solution of the residuein water was washed (×2) with chloroform and then evaporated. Theresidue was dissolved in methanol and treated with Amberlyst A21 baseresin until the solution was pH-7. The solids and solvent were removedand the residue was dissolved in water, treated with excess aqueous HCland then lyophilized. Trituration of the residue with ethanol gave(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol(7) hydrochloride salt as a white solid (25 mg). Recrystallised from 90%ethanol, the crystalline solid darkened but did not melt below 300° C.NMR (300 MHz, D₂O with Dcl, δ ppm): ¹³C (relative to internal acetone at33.2 ppm) 58.1 (C-1′), 61.4 (C-5′), 68.8 (C-4′), 73.3 (C-3′), 76.7(C-2′), 107.5 (q), 121.4 (q), 133.5 (C-2), 135.0 (q), 148.0 (C-6) and155.4 (q); ¹H (relative to internal acetone at 2.20 ppm), 3.90(H-4′),3.96 (m, H-5′,5″), 4.44 (dd, H-3′, J_(2′,3′) 5.4 Hz, J_(3′,4′)3.2 Hz), 4.71 (dd, J_(1′,2′) 9.0 Hz, H-2′), 5.00 (d, H-1′), 8.00 (s,H-6) and 9.04 (s, H-2).

Example 2 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolExample 2.1

[0255] A solution of(1S)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4dideoxy 1,4-imino-2,3-O-isopropylidene-D-ribitol (Example 1.5) (0.87 g)in ethanol was stirred with 10% Pd/C (100 mg) in an atmosphere ofhydrogen for 1.5 h. The solids and solvent were removed to give aresidue (0.61 g). To a solution of a portion of this residue (0.12 g) indichloromethane (10 ml) at 0° C. was added a solution of benzoylisothiocyanate in dichloromethane (31 mL in 1 ml). After 0.5 h thesolution was warmed to room temperature and1,8-diazabicyclo[5.4.0]undec-7-ene (80 mL) and methyl iodide (100 mL)were added. After another 0.5 h the reaction solution was applieddirectly to a silica gel column and elution afforded 0.16 g of(1S)-1-C-[3-(N-benzoyl-S-methylisothiocarbamoyl)amino-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.2

[0256] A solution of this S-methylisothiocarbamoylamino derivative,(0.20 g) in methanol saturated with ammonia was heated in a sealed tubeat 95° C. for 16 h. The solvent was removed and chromatography of theresidue afforded(1S)-1-C-[2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol.

Example 2.3

[0257] A solution of this protected iminoribitol (64 mg) intrifluoroacetic acid was allowed to stand at room temperature for 16 h.The solvent was removed and a solution of the residue in aqueousmethanol (1:1) was treated with Amberlyst A21 base resin until the pH ofthe solution was −7. The solids and solvent were removed and a solutionof the residue in water was treated with excess HCl and thenconcentrated to dryness. Trituration with ethanol gave(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt (24 mg), which darkened at ca. 260° C. but did notmelt below 300° C. NMR (300 MHz, D₂O with DCl, δ ppm): ¹³C (relative tointernal acetone at 33.1 ppm) 58.0 (C-1′), 61.4 (C-5′), 68.6 (C-4′),73.3 (C-3′) 76.3 (C-2′), 105.2 (q), 114.8 (q), 132.1 (C-6), 135.3 (q),153.4 (q) and 156.4 (q); ¹H (relative to internal acetone at 2.20 ppm)3.87 (m, H-4′), 3.94 (m, H-5′,5″), 4.40 (dd, J_(2′,3′) 5.0 Hz, J_(3′,4′)3.2 Hz, H-3′), 4.65 (dd, , J_(1′,2′) 9.1 Hz, H-2′), 4.86 (d, H-1′) and7.71 (s, H-6).

Examples 3-24

[0258] The following compounds may be prepared according co methodsdisclosed in the general description:

[0259] 3. (1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 1 using Method (H)

[0260] 4.(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 1 using Method (K).

[0261] 5.(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 1 using Method (K).

[0262] 6.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolmay be prepared from the product of Examples 1 or 2 using Method (M).

[0263] 7.(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 6 using Method (H).

[0264] 8.(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 6 using Method (K).

[0265] 9.(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 6 using Method (K).

[0266] 10.(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 2 by Method (H)

[0267] 11.(1S)-1-C-(2-amino-4-hydroxyppyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 2 by Method (K).

[0268] 12.(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 2 using Method (K).

[0269] 13.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitolmay be prepared by Methods (D), (E) and (F).

[0270] 14.(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 13 using Method (H)

[0271] 15.(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 13 using Method (K).

[0272] 16.(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 13 using Method (K).

[0273] 17.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitolmay be prepared from the product of Example 13 using Method (M)

[0274] 18.(1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 17 using Method (H).

[0275] 19.(1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 17 using Method (K).

[0276] 20.(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 17 using Method (K).

[0277] 21.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitolmay be prepared using a variation of Method (D) in which the compound ofFormula XIb or XIc is replaced by a corresponding compound in which thehydrogen atom in position 5 is replaced by protected amino group.

[0278] 22.(1R)-1-C-(5-amino-7-hydroyyrazolo[4,3-d]pyrimidin-3-yl)-14-imino-1,2,4-trideoxy-D-erythro-pentitolmay be prepared from the product of Example 21 using Method (H).

[0279] 23.(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitolmay be prepared from the product of Example 21 using Method (K).

[0280] 24. (1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitolmay be prepared from the product of Example 21 using Method (K)

Example 25 Enzyme Inhibition Results Example 25.1

[0281] Inhibition of purine nucleoside phosphorylases. Enzyme assayswere conducted to assess the effectiveness of the products of Examples 1and 2 (compounds Ib and Ic respectively) as inhibitors of purinenucleoside phosphorylase. The assays used human RBC and calf spleenpurine nucleoside phosphorylase (ex Sigma, 90% pure) with inosine assubstrate, in the presence of phosphate buffer, with detection ofreleased hypoxanthine using xanthine oxidase coupled reaction.

[0282] Materials. Inosine was obtained from Sigma. Xanthine oxidase (EC1.1.3.22, buttermilk), human erythrocyte (as a lyophilized powder) andbovine spleen (in 3.2 M ammonium sulfate) purine nucleosidephosphorylases (EC 2.4.2.1) were purchased from Sigma. Human purinenucleoside phosphorylases obtained as a powder was reconstituted in 100mM sodium phosphate buffer (pH 7.4) and rapidly frozen and stored at−80° C. Kinetic experiments were performed on a Uvikon 933 double beamultraviolet/visible spectrophotometer (Kontron Instruments, San Diego,Calif.).

[0283] Protein Concentrations. Protein concentrations for both isozymeswere determined based on the quantative ultraviolet absorbance, usingE_(1cm)1%=9.64 at 280 nm [Stoelkler et al, Biochemistry, 32 (1978) 278]and a monomer moleculer weight of 32,000 [Williams et al, Nucleic AcidsRes. 12 (1984) 5779].

[0284] Enzyme Assay. Enzymes were assayed spectrophotometrically usingthe coupled xanthine oxidase method [Kalckar, J. Biol. Chem. 167 (1947)429; Kim et al, J. Biol. Chem., 243 (1968) 1763]. Formation of uric acidwas monitored at 293 nm. A 40 mM inosine solution gave an absorbancechange of 0.523 units at 293 m, upon complete conversion of inosine touric acid and ribose 1-phosphate. Unless otherwise noted, the standardassay reaction contained: inosine (500 μM), potassium phosphate (50 mM,pH 7.5); xanthine oxidase (0.06 units) and purine nucleosidephosphorylase in a final volume of 1.0 mL.

[0285] One-Third-the-Sites Inhibition. Reaction mixtures of 6.7 nMbovine purine nucleoside phosphorylase containing varying amounts ofcompound Ib were pre-incubated at 30° C. for 1 hour. Reactions wereinitiated by addition of substrate (40 μM inosine, 3 times the K_(m)value) and assayed at 30° C. The reaction containing 0.6 nM inhibitor(concentration ratio of [compound Ib]/[purine nucleosidephosphorylase]=0.09) showed 29% inhibition, that containing 1 nMinhibitor ([compound Ib]/[purine nucleoside phosphorylase]=0.15) showed44%, whereas the reaction containing 3 nM inhibitor ([compoundIb]/[purine nucleoside phosphorylase]=0.44) had a rate decrease of 96%,and that containing 6 nM inhibitor ([compound Ib]/[purine nucleosidephosphorylate]=87%) showed 99% inhibition. These interactions are shownin FIG. 1.

[0286] Purine nucleoside phosphorylase is known to be a homotrimer witha catalytic site on each of the three protein subunits [Stoelkler et al,Biochemistry 32, (1978) 278]. When the concentration of enzyme subunitsis 6.7 nM, 50% inhibition of purine nucleoside phosphorylase occurs atapproximately 1.1 nM. This result demonstrates that compound Ib bindstightly and that binding of compound Ib to one site of the trimericenzyme leads to complete inhibition.

[0287] Activity Recovery from the Complex of Purine NucleosidePhosphorylase with Compound Ib. Purine nucleoside phosphorylase (6.7 μM)and sufficient compound Ib (3 μM) to inhibit 96% of purine nucleosidephosphorylase activity were incubated at 30° C. for 1 hour. An aliquotof this solution was diluted 1000-fold into a buffered solution of 500μM inosine containing xanthine oxidase (0.06 units). The production ofuric acid was monitored over time and the progres curve was fit to thekinetic model of FIG. 2.

[0288] Dilution of inhibited purine nucleoside phosphorylase into alarge volume of solution without inhibitor provided the rate of releaseof compound Ib from inhibited purine nucleoside phosphorylase. Underconditions of the experiment in FIG. 2, the time to achieve the newenzyme-inhibitor equilibrium is 5000 sec, an indication of a slow,tight-binding inhibitor [Morrison and Walsh, Advances Enzymol. 61 (1988)201]. The rate contant k₆ is an estimate of the apparent first-orderrate constant for dissociation of the complex under these experimentalconditions and is 2.9×10⁻⁴ sec⁻¹ in this example.

[0289] Inhibitory Mechanism. Slow, tight-binding inhibitors generallyfollow the kinetic mechanism [Morrison and Walsh, Advances Enzymol. 61(1988) 201]:

[0290] where EI is a rapidly formed, initial collision complex of purinenucleoside phosphorylase (E) and compound Ib (I) that slowly isomerizesto a tighter complex EI*. Product formation curves are described by thefollowing integrated rate equation 1:

P=v _(s) t+(v _(o) −v _(s))(1−e ^(−k t))/k  1

[0291] where P is the amount of product hypoxanthine (observed as uricacid in the present assay system), t is time, v_(o) is the initial rate,v_(s) is the final steady-state rate and k is the overall (observed)rate constant given by equation 2:

k=k6+k5[(I/K ₁)/(1+(S/K _(m))+(I/K _(i)))]  2

[0292] where K_(m) is the Michaelis complex for purine nucleosidephosphorylase, S is inosine concentration, I is the concentration ofcompound Ib and K_(i) is as described below.

[0293] The rate of formation of the tightly bound complex is k5 and therate of its dissociation is k6. K_(i), the inhibition constant forstandard competitive inhibition (which influences v_(o)) and K₁*, theoverall inhibition constant (which influences v_(s)), are defined as:

[0294] K_(i)=K4/k3

[0295] K_(i)*=K_(i)[k₆/(k₅+k₆)]

[0296] Determination of K_(i)*. K_(i)* was determined by measuring v_(s)for reactions at a range of inhibitor concentrations, plotting v_(s) vs[I] and fitting the curve to the competive inhibition equation 3:

v _(s) =V _(max) S/[K _(m)(1+I/K _(i)*)+S]  3

[0297] where V_(max) is the uninhibited reaction rate for purinenucleoside phosphorylase, and the remaining terms are described above.The result of this analysis indicates an overall effective inhibitionconstant (K_(i)*) of 2.5±0.2×10⁻¹¹ M (25±2 pM) for compound Ib (FIG. 3).

[0298] Approximation of K_(i), k₅ and k₆. Calculation of K₁ directlyfrom v_(o) and the competitive inhibition equation (above) is difficultfor compound Ib because v_(o) changes very little as a function of I atinhibitor concentrations which cause complete inhibition following slowonset. This result establishes that the initial dissociation constant K₁is much greater than the equilibrium dissociation constant K₁*.

[0299] Approximatiofts of k₅ and K₁ were calculated from k (valuesobtained from curve fits of equation 1, FIG. 4) by using equation 2.Using the knowledge that k₅<<k₅[(I/K_(i))/(1+(A/K_(m))+(I/K₁)], equation2 can be rearranged so that a double reciprocal plot of 1/k vs 1/[I]gives a straight line with y intercept=1/k₅ and x intercept of−(1/k₅)/[K₁/k₅)*(A/K_(m)))]. Substitution of these values into equation2 give an approximation for k₆. FIG. 4 demonstrates the slow-onset,tight-binding inhibition which occurs when a small concentration ofenzyme (0.8 nM) competes for 200 nM compound Ib in the presence of 500μM inosine. Under these conditions the apparent first order rateconstant for onset of inhibition in FIG. 4 was 26×10⁻⁴ sec⁻¹.

[0300] The result of FIG. 4 demonstrates that even at inosineconcentrations over 100 times that present in human serum or tissues,compound Ib can give 99% inhibition of the enzyme after several minutesof slow-onset inhibition. Based on analyses of experiments of the typeshown in FIGS. 1-4, the experimentally estimated dissociation constantsand rates for the bovine purine nucleoside phosphorylase with compoundIb are:

[0301] K_(m)=15 μM

[0302] K_(i)=19±4 nM

[0303] K_(i)*=25±2 pM

[0304] k₅=1.4±0.2×10⁻² sec⁻¹

[0305] k₆=1.8±0.5×10⁻⁵ sec−1

[0306] Inhibition of Human Purine Nucleoside Phosphorylase. Studiessimilar to those described above for the interaction of bovine purinenucleoside phosphorylase were conducted with purine nucleosidephosphorylase (PNP) from human erythrocytes. The values for the overallinhibition constant, K_(i)*, for the interaction of human and bovine PNPwith compound Ib are: enzyme K_(i)*, compound Ib K_(i)*, compound Ichuman PNP 72 ± 26 pM 29 ± 8 pM bovine PNP  23 ± 5 pM 30 ± 6 pM

[0307] The compound Ic is a more efficient inhibitor for the humanenzyme than compound Ib, but compound Ib is slightly more efficient atinhibiting the bovine enzyme. Compounds Ib and Ic are more efficient atinhibiting both PNP enzymes than previously reported compounds.

[0308] Summary of Compounds Ib and Ic as Inhibitors of Purine NucleosidePhosphorylases. Inhibitors usually function by binding at everycatalytic site to cause functional inhibition in living organisms. Theone-third-the-sites inhibition and the slow-onset tight-bindinginhibition described above indicate that compounds Ib and Ic are verypotent inhibitors of purine nucleoside phosphorylases able to functionin the presence of a large excess of substrate.

[0309] The methods for the determination of the kinetic constants aregiven in detail in Merkler, D. J., Brenowitz, M., and Schramm, V. L.Biochemistry ,9 (1990) 8358-8364.

Example 25.2

[0310] Oral Availability and in vivo Efficacy of Compound Ib as a PNPInhibitor. A single oral dose of 10⁻⁷ mole of Ccmpound Ib (27 μg) wasadministered with food to a young adult male mouse. Blood samples werecollected from the tail at times indicated in FIG. 5. Dilution of bloodinto saline containing 0.2% Triton X-100 (final concentration 0.15%)resulted in lysis of blood cells and release of enzyme. PNP activity wasmeasured with inosine and phosphate as substrates as indicated above.The results establish that Compound Ib is absorbed into the blood andtaken up by blood cells to cause PNP inhibition with a half-time (t_(½))of 14 minutes. Blood samples were taken for an extended time andanalyzed for PNP activity to determine the biological t_(½) for CompoundIb for inhibitors of blood PNP. The activity of blood PNP recovered witha t_(½) of 100 hours. These results establish that Compound Ib is orallyavailable and has an extended period of biological effectiveness. Thesetests establish that the compounds described herein have favorablepharmacological lifetimes.

[0311] Inhibition of Protozan Nucleoside Hydrolases by Compounds Ib andIc. Protozan parasites use the hydrolysis of purine nucleosides such asinosine to provide purine bases such as hypoxanthine to provideessential precursors for RNA and DNA synthesis. Protozoan parasites arepurine auxotrophs. Using inhibition methods similar to those describedabove, a nucleoside hydrolase from Crithidia fasciculata [Parkin, et al,J Biol, Chem. 266 (1991) 20658] and a nucleoside hydrolase fromTrypanosoma brucei brucei [Parkin, J. Biol. Chem. (1996) 21713] weretested for inhibition by compounds Ib and Ic. The inhibition ofnucleoside hydrolase from C. fasciculata by Compound Ib is exemplifiedin FIG. 6. Similar studies indicated that Coumpound Ib and Ic arenanamolar inhibitors for nucleoside hydrolases from C. fasciculata andfrom T. brucei brucei. Compound Ic (A═CH, B═NH₂, D═H, X═OH, Y═H, Z═OH)is a nanamolar inhibitor of both enzymes and Compound Va (OR═NH₂, z′═OH,CO₂Bu═H or H₂, and the isopropylidine group removed to form two hydroxylgroups) is also a nanamolar inhibitor of both enzymes. The results aresummarised below. K_(i) Values (nM) Compound Compound Compound Compoundenzyme source Ia^(a) Ib^(b) Ic^(b) Va^(b) nucleoside 42 ± 2 nM  40 nM  7 nM  3 nM hydrolase C. fasciculata nuceloside 24 ± 3 nM 108 nM 0.9 nM23 nM hydrolase T. brucei brucei

[0312] The inhibitors bind in direct competition with substrate,therefore the K₁ inhibition constants are direct competitive inhibitionvalues. The compounds provide sufficient inhibition to the purinenucleoside hydrolases to inhibit protozoan parasites at readilyaccessible pharmacological doses.

[0313] The methods and materials used are as described in published PCTinternational application WO 97/31008 using p-nitrophenyl riboside assubstrate.

Example 25.3

[0314] Inhibition of Purine Phosphoribisyl Transferases (PPRT) by5′-Phosphates of Compounds Ib and Ic. Protozoan parasites, human tissuesand tumors use PPRT for salvage of purine bases. Interruption of PPRTactivity is expected to disrupt purine metabolism in these systems.5′-phosphorylated Compounds I and Ic were anlyzed for inhibition of PPRTfrom human and malarial origins. The slow-onset inhibition curve for the5′-phosphate of Compound Ib with malaria PPRT is illustrated in FIG. 7.The K₁* determination for the 5′-phosphate of Compound Ib with malarialPPRT is shown in FIG. 8. Analysis of both human and malarial enzymeswith the 5′-phosphates of Compounds Ib and Ic are summarized below.enzyme Compound Ib-5′-phosphate Compound Ic-5′-phosphate source K_(i)K_(i)* K_(i) K_(i)* PPRT human 40 nM 3 nM 14 nM 8 nM PPRT 33 nM 3 nM 48nM slow onset malaria not observed

[0315] Full inhibition studies indicated that the inhibitors arecompetitive with IMP. The nanamolar inhibition constants for bothinhibitors with both enzymes are readily accessible pharmacologic dosesof these inhibitors. It is anticipated that the nucleoside kinaseactivities of human and/or parasitic organisms will convert one or moreof the compounds described herein to the respective 5′-phosphates. Thesecompounds thereby provide precursors for pharmacologic doses of the5′-phosphates for intracellular interruption of PPRT activity. Thecellular uptake of Compounds I and Ic have been documented with mice andwith human red cells.

Example 26 Tablet

[0316] 4 grams of the product of Example 1 is mixed with 96 grams oflactose and 96 grams of starch. After screening and mixing with 2 gramsof magnesium stearate, the mixture is compressed to give 250 milligramtablets.

Example 27 Gelatin Capsule

[0317] Ten grams of the product of Example 1 is finely ground and mixedwith 5 grams of talc and 85 grams of finely ground lactose. The powderis filled into hard gelatin capsules.

Example 28 Preparation of(1R)-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-erythro-pentitolExample 28.1

[0318] A solution of(1S)-5-O-tert-butyldimethylsilyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1.93 g) in trifluoroacetic acid (20 ml) was allowed to stand at roomtemperature overnight. The solution was concentrated and a solution ofthe residue in water was washed (×2) with chloroform and then evaporatedto afford (1S)-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-D-ribitol (1.0 g)as the trifluoroacetic acid salt.

Example 28.2

[0319] A solution of the crude product from Example 3.1 (1.0 g) inmethanol (20 ml) containing di-tert-butyldicarbonate (2.09 g) wasadjusted to neutral pH by the addition of triethylamine and stirred atroom temperature for 16 h. The solution was concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-D-ribitol(0.80 g).

Example 28.3

[0320] 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (0.9 ml) was addeddropwise to a solution of the product from Example 3.2 (0.8 g) andimidazole (0.70 g) in N,N-dimethylformamide (10 ml) at 0° C. Theresulting solution was allowed to warm to room temperature, diluted withtoluene, washed with water (×3), dried, concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanamethyl-1,4-dideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(1.4 g).

Example 28.4

[0321] A solution of the product from Example 3.3 (1.5 g) in toluene (20ml) containing thiocarbonyldiimidazole (0.9 g) was stirred at 90° C. for2 h. The solution was concentrated and then chromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-2-O-[imidazole(thiocarbonyl)]-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(1.8 g).

Example 28.5

[0322] To a solution of the product from Example 28.4 (1.8 g) in toluene(50 ml) was added tri-n-butyltin hydride (1.0 ml) and the solution washeated at 80° C. for 3 h. The solution was concentrated and thenchromatography afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.74 g).

Example 28.6

[0323] To a solution of the product from Example 3.5 (0.74 g) inN,N-dimethylformamide (10 ml) was addedtert-butoxy-bis(dimethylamino)methane (1.5 ml) and the solution washeated at 65-70° C. for 1 h. Toluene (20 ml) was added and the solutionwas washed (×3) with water, dried and concentrated to dryness. Theresidue was dissolved in tetrahydrofuran/acetic acid/water (1:1:1 v/v/v,40 ml) at room temperature. After 1.5 h, chloroform (50 ml) was addedand the mixture was washed with water (×2), aqueous sodium bicarbonate,and then dried and evaporated to dryness. Chromatography of the residuegave(1R)-N-tert-butoxycarbonyl-1-C-(1-cyano-2-hydroxyethenyl)-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.68 g).

Example 28.7

[0324] Glycine hydrochloride ethyl ester (0.90 g) and sodium acetate(1.0 g) were added to a stirred solution of the product from Example 3.6(0.68 g) in methanol (10 ml). The mixture was stirred at roomtemperature for 16 h and then concentrated to dryness. Chromatography ofthe residue gave the(1R)-N-tert-butoxycarbonyl-1-C-[1-cyano-2-(ethoxycarbonylmethylamino)ethenyl]-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.80 g) as a diastereomeric mixture.

Example 28.8

[0325] A solution of the product from Example 3.7 (0.80 g) in drydichloromethane (20 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(3.6 ml) and benzyl chloroformate (1.7 ml) was heated under refluxovernight, then cooled and washed with dilute aqueous HCl and thenaqueous sodium bicarbonate, dried and concentrated. Chromatography ofthe residue afforded(1R)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(0.70 g).

Example 28.9

[0326] A solution of the product from Example 28.8 (0.28 g) in ethanol(10 ml) was stirred with formamidine acetate (0.50 g) under reflux for 8h. The solvent was removed and chromatography of the residue gave(1R)-N-tert-butoxycarbonyl-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxa-1,3-diyl)-D-erythro-pentitol(120 mg).

Example 28.10

[0327] A solution of the product from Example 28.9 (120 mg) intrifluoroacetic acid (2 ml) was allowed to stand at room temperatureovernight. The solution was concentrated and a solution of the residuein water was washed (×2) with chloroform and then evaporated. Theresidue was dissolved in tetrahydrofuran and treated withtetrabutylammonium fluoride trihydrate (200 mg) and stirred for 1 h. Thesolvent was evaporated and chromatography gave a residue which wasredissolved in methanolic HCl. The resulting precipitate was filtered toafford(1R)-1,2,4-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-erthro-pentitolhydrochloride salt as a white solid (17 mg) which darkened but did notmelt below 300° C. NMR (300 MHz, D₂O, d ppm): ¹³C 38.8 (C-2′), 53.4(C-1′), 59.3 (C-5′), 69.1 (C-4′), 71.5 (C-3′), 107.6 (q), 118.6 (q),130.4 (C-2), 135.9 (q), 144.6 (C-6), and 153.7 (q); ¹H 2.69 (dd, J 14.3Hz, J 6.4 Hz, H-2′), 2.60 (ddd, J 14.3 Hz, J 12.2 Hz, J 5.7 Hz, H-2″)3.87 (m, 3H, H-4′, H-5′), 4.57 (m ¹H, H-3′), 5.26 (dd, 1H, J 12.1 Hz, J6.4 Hz, H-1′), 7.80 (s, H-6) and 8.65 (s, H-2). HRMS (MH⁺) calc. forC₁₁H₁₄N₄O₃: 251.1144; found: 251.1143.

Example 29 Preparation of(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,2,4-trideoxy-1,4-imino-D-erythro-pentitolExample 29.1

[0328] A solution of(1R)-1-C-[3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol(Example 28.8) (0.78 g) in ethanol (10 ml) was stirred with 10% Pd/C(100 mg) in an atmosphere of hydrogen for 1.5 h. The solids and solventwere removed to give a residue (0.62 g). To a solution of this residuein dichloromethane (10 ml) at 0° C. was added a solution (4.8 ml) ofbenzoyl isothiocyanate in dichloromethane (0.30 ml in 10 ml). After 0.5h, the solution was warmed to room temperature and1,8-diazabicyclo[5.4.0]undec-7-ene (0.32 ml) and methyl iodide (0.70 ml)were added. After another 0.5 h the reaction solution was applieddirectly to a silica gel column and elution afforded 0.67 g of(1R)-1-C-[3-(1-benzamido-1-methylthiomethyleneamino)-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol.

Example 29.2

[0329] A solution of the product from Example 29.1 (0.67 g) in methanolsaturated with ammonia (20 ml) was heated in a sealed tube at 105° C.for 16 h. The solvent was removed and chromatography of the residueafforded (1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,2,4-trideoxy-1,4-imino-3,5-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-D-erythro-pentitol (0.30 g).

Example 29.3

[0330] A solution of the product from Example 29.2 (300 mg) intrifluoroacetic acid (5 ml) was allowed to stand at room temperature for16 h. The solvent was removed and the residue was dissolved intetrahydrofuran, treated with tetrabutylammonium fluoride trihydrate(200 mg) and stirred for 1 h. The solvent was removed and the residuewas dissolved in methanol (5.0 ml) and acetyl chloride (0.75 ml) wasadded dropwise and the reaction allowed to stand at room temperature for16 h. The reaction was diluted with ether (25 ml) and the resultingcrystals were filtered to afford(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,2,4-trideoxy-1,4-imino-D-erythro-pentitolhydrochloride salt (89 mg), which did not melt below 300° C. NMR (300MHz, D₂O d ppm): ¹³C 38.8 (C-2′) 53.4 (C-1′), 59.3 (C-5′), 69.1 (C-4′),71.5 (C-3′), 107.6 (q), 118.6 (q), 130.4 (C-2), 135.9 (q), 144.6 (C-6),and 153.7 (q); ¹H 2.69 (dd, 1H, J 14.3 Hz, J 6.3 Hz, H-2′), 2.63 (ddd,1H, J 14.1 Hz, J 12.3 Hz, J 5.7 Hz, H-2 ″) 3.88 (m, 3H, H-4′, H-5′),4.55 (m, 1H, H-3′), 5.14 (dd, 1H, J 12.2 Hz, J 6.3 Hz, H-1′), and 7.63(s, H-6).

Example 30 Preparation of(1S)-1,4,5-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol hydrochloride salt Example 30.1

[0331] A solution of the product from Example 1.5 (0.45 g) indichloromethane (10 ml) was treated with triethylamine (0.45 ml),4-dimethylaminopyridine (20 mg) and then methanesulfonyl chloride (0.1ml). The solution was stirred for 1 h and then washed with 2M aq HCl, aqbicarbonate and processed conventionally. The crude product wasdissolved in toluene (10 ml) containing tetrabutylammonium bromide (1.55g) and the solution was heated at 100° C. for 2 h. The cooled solutionwas washed with water, and processed to give, after chromatography,(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-5-bromo-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.27 g).

Example 30.2

[0332] A solution of the product from Example 30.1 (0.27 g) in ethanol(10 ml) containing triethylamine (0.19 ml) was stirred with 20%Pd(OH)₂/C (0.1 g) in a hydrogen atmosphere for 16 h. The solids andsolvent were removed and chromatography afforded(1S)-1-C-(3-amino-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.15 g).

Example 30.3

[0333] A solution of the product from Example 30.2 (75 mg) in ethanolcontaining formamidine acetate (0.15 g) was heated under reflux for 4 h.The solvent was removed and chromatography afforded(1S)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1-C-[4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl]-1,4-imino-2,3-O-isopropylidene-D-ribitol(69 mg).

Example 30.4

[0334] The product from Example 30.3 (69 mg) was dissolved intrifluoroacetic acid (5 ml) and the solution was allowed to stand atroom temperature for 16 h. The solvent was removed and a solution of theresidue in 50% aqueous methanol (10 ml) was treated with Amberlyst A21base resin until the pH was −7. The solids and solvent were removed andthe residue was treated with excess aqueous HCl and lyophilized to give(1S)-1,4,5-trideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol hydrochloride salt (46 mg). ¹³C NMR (75 MHz, D₂Owith DCl, d ppm): 155.6 (C), 147.1 (CH), 137.4 (C), 132.6 (CH), 121.0(C), 108.2 (C), 76.5 (C-3), 75.6 (C-2), 63.2 (C-4), 58.2 (C-1), 18.1(C-5).

Example 31 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4,5-trideoxy-1,4-imino-D-ribitolhydrochloride salt Example 31.1

[0335] A solution of ben7oyl isothiocyanate (0.33 ml of 0.4 ml in 5 mlof dichloromethane) was added to the product from Example 5.2 (75 mg) indichloromethane (5 ml) at 0° C. After 1 h,1,8-diazabicyclo[5.4.0]undec-7-ene (0.06 ml) and methyl iodide (0.1 ml)were added and the solution was stirred at room temperature for 1 h.Chromatography then afforded1(S)-1-C-[3-(1-benzamido-1-methylthio-methyleneamino)-2-ethoxycarbonyl-4-pyrrolyl]-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.10 g). A solution of this material in methanol (5 ml) saturated withammonia was heated in a sealed tube at 95° C. for 16 h and thenevaporated. Chromatography afforded (1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(28 mg).

Example 31.2

[0336] The product from Example 31.1 (28 mg) was treated as for Example30.4 above to give(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4,5-trideoxy-1,4-imino-D-ribitolhydrochloride salt (16 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 156.5(C), 153.5 (C), 135.8 (C), 131.7 (CH), 114.9 (C), 105.6 (C), 76.7 (C-3),75.7 (C-2), 63.4 (C-4), 58.1 (C-1), 18.4 (C-5).

Example 32 Preparation of(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt Example 32.1

[0337] A solution of the product from Example 1.3 (0.15 g) in methanol(5 ml) containing aminoacetonitrile (0.12 g) and sodium acetate (0.20 g)was heated under reflux for 4 h and then concentrated. Chromatographyafforded1(S)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1-C-[1-cyano-2-cyanomethylamino-ethenyl]-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.12 g) as a diastereomeric mixture. A solution of this material indichloromethane (10 ml) containing 1,8-diazabicyclo[5.4.0]undec-7-ene(0.7 ml) and benzyl chloroformate (0.33 ml) was heated under reflux for1 h. Conventional processing and chromatography afforded(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-cyano-4-pyrrolyl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.125 g).

Example 32.2

[0338] A solution of the product from Example 32.1 (0.125 g) in ethanol(10 ml) was stirred in an atmosphere of hydrogen with 10% Pd/C (20 mg)for 0.5 h. The solids were removed, formamidine acetate (0.21 g) wasadded to the filtrate and the solution was heated under reflux for 16 hand then concentrated. Chromatography of the residue gave(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(80 mg).

Example 32.3

[0339] The product from Example 32.2 (80 mg) was treated as for Example30.4 above to give(1S)-1-C-(4-aminopyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitolhydrochloride salt (35 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 152.1(C), 146.2 (CH), 140.7 (C), 135.3 (CH), 115.4 (C), 107.7 (C), 76.0(C-2), 73.1 (C-3), 68.4 (C-4), 61.3 (C-5), 58.3 (C-1).

Example 33 Preparation of(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol5-phosphate bis-ammonium salt

[0340] The product from Example 2.2 (0.13 g) in dry acetonitrile (6 ml)containing tetrazole (0.105 g) was stirred at room temperature whileN,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine was addedslowly dropwise until t.l.c. indicated complete reaction, thenmeta-chloroperbenzoic acid (60 mg) was added followed by further smallquantities of the oxidant until t.l.c. indicated the initial product wasfully reacted. Chloroform was added and the solution was washed withaqueous sodium bicarbonate, dried and concentrated. Chromatographyafforded the phosphate ester (190 mg) which was stirred in ethanol (10ml) in an atmosphere of hydrogen with 10% Pd/C (80 mg) for 1 h. Thesolids and solvent were removed and the residue was dissolved intrifluoroacetic acid (5 ml) and allowed to stand at room temperature for16 h. The solution was concentrated by evaporation and the residue inwater was applied to a column of Amberlyst A15 acid resin. The columnwas washed with water and then with 2M aqueous ammonia to elute theproduct. Concentration and trituration of the residue with waterafforded(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol5-phosphate bis-ammonium salt (50 mg), referred to as the 5′-phosphateof compound Ib. ¹³C NMR (75 MHz, TFA-D, d ppm): 146.9 (C), 144.0 (C),127.0 (C), 124.5 (CH), 105.1 (C), 95.6 (C), 66.3 (CH), 64.0 (CH), 59.2(CH), 56.2 (CH₂) 50.2 (CH).

Example 34 Preparation of(1S)-1,4,5-trideoxy-5-fluoro-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt Example 34.1

[0341] To a solution of the product from Example 1.2 (1.48 g) intetrahydrofuran (10 ml) was added tetrabutylammonium fluoride (6 ml, 1Min THF). After 2 h the solution was evaporated and chromatography of theresidue afforded(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(1.15 g). A solution of 0.84 g of this material in dichloromethane (20ml) containing triethylamine (1.0 ml) was stirred whilediethylaminosulfur trifluoride (0.36 ml) was added. After 2 h, methanol(1 ml) was added and the solution was evaporated. Chromatography gave(1S)-N-tert-butoxycarbonyl-1-C-cyanomethyl-1,4,5-trideoxy-5-fluoro)-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.36 g).

Example 34.2

[0342] The product from Example 34.1 (0.36 g) was treated in the samemanner as described for examples 1.3 and then 1.4 and 1.5 above to give(1S)-1-C-(3-amino-1-N-benzyloxycarbonyl-2-ethoxycarbonyl-4-pyrrolyl)-N-tert-butoxycarbonyl-1,4,5-trideoxy-5-fluoro-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.23 g).

Example 34.3

[0343] The product from Example 34.2 (0.12 g) was treated as describedfor examples 1.6 and then 1.7 above to give, after lypohilization,(1S)-1,4,5-trideoxy-5-fluoro-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride salt (43 mg). ¹³C NMR (75 MHz, D₂O with DCl, d ppm): 146.8(CH), 132.6 (CH), 83.0 (J_(C,F) 169 Hz, C-5), 76.1 (C-2), 72.7 (C-3),66.4 (J_(C,F) 18 Hz, C-4), 59.0 (C-1).

Example 35(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitolExample 35.1

[0344] Hydrogen peroxide (0.5 ml) was added dropwise to a solution ofthe product from Example 32.1 (90 mg) and potassium carbonate (50 mg) indimethylsulfoxide (1.0 ml). The reaction was stirred for 10 minutes,diluted with water (50 ml), extracted with ethyl acetate (3×20 ml), andthe combined organic layers dried and concentrated. Chromatography ofthe resulting residue afforded(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-N-tert-butoxycarbonyl-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(20 mg).

Example 35.2

[0345] A solution of the product from Example 35.1 (20 mg) intrifluoroacetic acid (1 ml) was allowed to stand at room temperature for16 h. The solvent was removed and the residue in water (20 ml) waswashed with dichloromethane (2×5 ml). The aqueous layer was evaporatedand chromatography afforded(1S)-1-C-(3-amino-2-carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitol(10 mg). NMR (300 MHz, D₂O): ¹³C 59.3 (C-4′), 64.0 (C-5′), 67.7 (C-1′),74.4 (C-3′), 77.6 (C-2′), 113.2 (q), 124.1 (C-5), 126.2 (q), 141.0 (q),and 168.7 (q). HRMS (MH⁺) calc. for C₁₀H₁₇N₄O₄: 257.12498; found:257.12535.

Example 36 Preparation of(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo-[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitolExample 36.1

[0346] 2,4-Dihydroxy-6-methyl-5-nitropyrimidine (G. N. Mitchell and R.L. McKee, J. Org. Chem., 1974, 39, 176-179) (20 g) was suspended inphosphoryl chloride (200 ml) containing N,N-diethylaniline (20 ml) andthe mixture was heated under reflux for 2 h. The black solution wasconcentrated to dryness and the residue was partitioned between water(600 ml) and ether (150 ml). The aqueous phase was further extractedwith ether (150 ml) and the combined organic phases were washed withaqueous sodium bicarbonate and processed conventionally to give2,4-dichloro-6-methyl-5-nitropyrimidine (23.1 g).

Example 36.2

[0347] To a solution of the product of Example 36.1 (17 g) in benzylalcohol (80 ml) was added a 1.1 M solution of sodium benzylate in benzylalcohol (199 ml). After 1 h at room temperature, ether (500 ml) wasadded and the solution was washed with water. The organic phase wasdried and concentrated to dryness under high vacuum. The crude residuein dry N,N-dimethylformamide (100 ml) and N,N-dimethylformamide dimethylacetal (25 ml) was heated at 100° C. for 3 h and then the solution wasconcentrated to dryness. Trituration of the residue with ethanol andfiltration afforded2,4-dibenzyloxy-6-(2-dimethylaminovinyl)-5-nitropyrimidine as an orangesolid (24.5 g).

Example 36.3

[0348] Zinc dust (30 g) was added to a solution of the product fromExample 36.2 (20 g) in acetic acid (300 ml) with cooling to control theexotherm. The resulting mixture was then stirred for 2 h, filtered, andthe filtrate was concentrated to dryness. The residue was partionedbetween chloroform and aqueous sodium bicarbonate, the organic layer wasdried and then concentrated to dryness to give a solid residue of2,4-dibenzyloxypyrrolo[3,2-d]pyrimidine (15.2 g).

Example 36.4

[0349] Sodium hydride (0.5 g, 60% dispersion in oil) was added to asolution of the product from example 36.3 (2.0 g) in tetrahydrofuran (40ml) followed by tert-butyldimethylsilyl chloride (1.37 g) and themixture was stirred for 1 h. The reaction was quenched with dropwiseaddition of water and then partitioned between ether (100 ml) and water(150 ml). The organic phase was dried and concentrated to dryness. Asolution of the residue in dichloromethane (40 ml) was stirred whileN-bromosuccinimide added slowly poriton-wise until t.l.c. analysisindicated complete conversion to a less polar product. The solution waswashed with water, aqueous sodium bicarbonate, dried and concentrated.Chromatography of the residue afforded2,4-dibenzyloxy-7-bromo-9-N-tert-butyldimethylsilylpyrrolo[3,2-d]pyrimidineas a white solid (1.8 g).

Example 36.5

[0350] An imine was prepared from5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.30 g) by N-chlorination with N-chlorosuccinimide followed byelimination of hydrogen chloride with lithium tetramethylpiperidide asdescribed in Example 1.1, but with the following modifications: (i) whenaddition of the solution of lithium tetramethylpiperidide was complete,petroleum ether was added and the solution was washed with water, driedand concentrated to dryness; (ii) the residue was chromatographed onsilica gel eluted with 0.2% triethylamine and 30% ethyl acetate inhexanes to afford the pure imine (0.215 g). A solution of this imine inether (2 ml) was added to a solution prepared by slow addition ofbutyllithium (1.4 M in hexanes) to a solution of the product fromExample 36.4 (0.786 g) in anisole (20 ml) and ether (30 ml) at −70° C.until t.l.c. analysis indicated lithium exchange with the startingmaterial was complete. The mixture was allowed to slowly warm to −15°C., and then was washed with water, dried and concentrated.Chromatography of the residue afforded(1S)-1-C-)2,4-dibenzyloxy-9-N-tert-butyldimethylsilylpyrrolo[3,2-d]pyrimidin-7-yl)-5-O-tert-butyldimethylsilyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(0.225 g).

Example 36.6

[0351] A solution of the product from Example 36.5 (0.10 g) in ethanol(5 ml) was stirred in a hydrogen atmosphere with 10% palladium oncharcoal (0.05 g) for 2 h. The solids and solvent were removed andconcentrated aqueous hydrochloric acid (1 ml) was added to a solution ofthe residue in methanol (5 ml). After standing overnight, the solutionwas concentrated to dryness and the residue was extracted with ether andthen triturated with ethanol and filtered to give(1S)-1,4-dideoxy-1-C-)2,4-dihydroxypyrrolo[3,2-d][pyrimidin-7-yl)-1,4-imino-D-ribitolhydrochloride (0.025 g). ¹³C NMR (D₂O), δ (ppm): 159.8 (C), 155.8 (C),137.1 (C), 131.4 (CH), 114.2 (C), 104.1 (C), 76.2 (CH), 73.7 (CH), 68.5(CH), 61.6 (CH₂) and 58.5 (CH).

Example 37 - Preparation of1,4-dideoxy-(1S)-1-C-(2,4-dihydroxypyrrolo-[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol 5-phosphate bis-ammonium salt Example 37.1

[0352] A solution tetrabutylammonium fluoride (1 M, 0.5 ml) was added toa solution of the bis-silylated product from Example 36.5 (110 mg) intetrahydrofuran. After 2 h, the solution was diluted with toluene,washed with water (×2), dried, and evaporated to dryness. The resultingsyrup was dissolved in methanol and tert-butoxycarbonic anhydride (65mg) was added. After 30 min, the reaction mixture was concentrated todryness and subjected to chromatography to give(1S)-1-C-(2,4-dibenzyloxypyrrolo[3,2-d]pyrimidin-7-yl)-N-tert-butoxycarbonyl-1,4-dideoxy-1,4-imino-2,3-O-isopropylidene-D-ribitol(64 mg).

Example 37.2

[0353] The product for Example 37.2 (64 mg) was converted by the methoddetailed in Example 33 into,1,4-dideoxy-(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol5-phosphate bis-ammonium salt (11 mg); ¹³C-NMR (D₂O), δ (ppm): 156.0(C), 151.9 (C), 134.0 (C), 127.3 (CH), 110.9 (C), 102.8 (C), 75.1 (CH),70.4 (CH), 65.1 (CH), 61.9 (CH₂), and 54.5 (CH).

[0354] Aspects of the invention have been described by way of exampleonly and it should be appreciated that modifications and additionsthereto may be made without departing from the scope of the invention.

What is claimed is:
 1. A compound having the formula:

wherein A is CH or N; B is chosen from OH, NH₂, NHR, H or halogen; D ischosen from OH, NH₂, NHR, H, halogen or SCH₃; R is an optionallysubstituted alkyl, aralkyl or aryl group; and X and Y are independentlyselected from H, OH or halogen except that when one of X and Y ishydroxy or halogen, the other is hydrogen; and Z is OH or, when X ishydroxy, Z is selected from hydrogen, halogen, hydroxy, SQ or OQ where Qis an optionally substituted alkyl, aralkyl or aryl group; or a tautomerthereof; or a pharmaceutically acceptable salt thereof; or an esterthereof; or a prodrug thereof.
 2. The compound of claim 1, wherein oneof B and/or D is NHR, and R is C₁₋C₄ alkyl.
 3. The compound of claim 1,wherein either D is H, or B is OH, or both.
 4. The compound of claim 1,wherein B is OH, D is H, OH or NH₂, X is OH or H, Y is H.
 5. Thecompound of claim 4, wherein Z is OH, H or methylthio.
 6. The compoundof claim 5, wherein Z is OH.
 7. The compound of claim 1 selected from(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol,(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol,(1R)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol,(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol,(1R)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(2,4-dihydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-5-methylthio-D-ribitol,(1R)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1-C-(2-amino-4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol,(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol,(1R)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol,(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-D-ribitol,(1R)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1,4-dideoxy-1-C-(5,7-dihydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-5-methylthio-D-ribitol,(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-D-ribitol,(1R)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,2,4-trideoxy-D-erythro-pentitol,(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-imino-1,4,5-trideoxy-D-ribitol,(1S)-1-C-(5-amino-7-hydroxypyrazolo[4,3-d]pyrimidin-3-yl)-1,4-dideoxy-1,4-imino-5-methylthio-D-ribitol,or a tautomer thereof; or a pharmaceutically acceptable salt thereof. 8.The compound of claim 1 which is (1S)-1,4-dideoxy-1-C-(4-hydroxyoyrrolo[3,2-d]pyrimidin-7-yl)-1,4-imino-D-ribitol, or tautomerthereof, or a pharmaceutically acceptable salt thereof.
 9. The compoundof claim 1 which is(1S)-1-O-(2-amino-4-hydroxyoyrro[3,2-d]pyrimidnn-7-yl)-1,4-dideoxy-1,4-imino-D-ribitol, or tautomer thereof, or apharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 1 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 11.A pharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 2 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 12. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 3effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 13. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 4 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 14.A pharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 5 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 15. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 6effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 16. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 7 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 17.A pharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 8 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 18. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 9effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 19. A pharmaceuticalcomposition for treatment and/or prophylaxis of a protozoan infectioncomprising an amount of a compound of claim 1 effective for inhibitingat least one parasite purine nucleoside hydrolase, purine nucleosidephosphorylase and/or purine phosphoribosyl transferase and apharmaceutically acceptable carrier diluent.
 20. A method for decreasingT-cell function in a mammal comprising administering to the mammal acompound of claim 1, whereby said compound inhibits purine nucleosidephosphorylase.
 21. A method for treatment and/or prophylaxis of aninfection caused by protozoan parasite comprising administering to asubject an amount of a compound of claim 1 effective to inhibit at leastpurine nucleoside hydrolase, purine nucleoside phosphorylase, and/orpurine phosphoribosyl transferase.
 22. A method for killing parasitescomprising administering the parasite an amount of a compound of claim 1effective for inhibiting at least one purine nucleoside hydrolase,purine nucleoside phosphorylase, and/or purine phosphoribosyltransferase.
 23. A compound having the formula:

wherein A is CH or N; X and Y are independently selected from H, OH orhalogen except that when one of X and Y is hydroxy or halogen, the otheris hydrogen; and Z is OH or, when X is hydroxy, Z is selected fromhydrogen, halogen, hydroxy, SQ or OQ where Q is an optionallysubstituted alkyl, aralkyl or aryl group; E is chosen from CO₂H or acorresponding salt form, CO₂R, CN, CONH₂, CONHR or CONR₂; and G ischosen from NH₂, NHCOR, NHCONHR or NHCSNHR; or a tautomer thereof; or apharmaceutically acceptable salt thereof; or an ester thereof; or aprodrug thereof.
 24. The compound of claim 23, wherein E is CONH₂ and Gis NH₂.
 25. The compound of claim 23, wherein E is CONH₂, G is NH₂, X isOH or H, Y is H.
 26. The compound of claim 23 wherein Z is OH, H ormethylthio.
 27. The compound of claim 26, wherein Z is OH.
 28. Thecompound of claim 23 which is (1S)-1-C-(3-amino-2carboxamido-4-pyrrolyl)-1,4-dideoxy-1,4-imino-D-ribitol, or tautomerthereof, or a pharmaceutically acceptable salt thereof.
 29. Apharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 23 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 30. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 24effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 31. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 25 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 32.A pharmaceutical composition for the suppression of T-cell functioncomprising an amount of a compound of claim 26 effective for inhibitingpurine nucleoside phosphorylase, and a pharmaceutically acceptablecarrier or diluent.
 33. A pharmaceutical composition for the suppressionof T-cell function comprising an amount of a compound of claim 27effective for inhibiting purine nucleoside phosphorylase, and apharmaceutically acceptable carrier or diluent.
 34. A pharmaceuticalcomposition for the suppression of T-cell function comprising an amountof a compound of claim 28 effective for inhibiting purine nucleosidephosphorylase, and a pharmaceutically acceptable carrier or diluent. 35.A method for decreasing T-cell function in a mammal comprisingadministering to the mammal a compound of claim 23, whereby saidcompound inhibits purine nucleoside phosphorylase.
 36. A method fortreatment and/or prophylaxis of an infection caused by protozoanparasite comprising administering to a subject an amount of a compoundof claim 23 effective to inhibit at least purine nucleoside hydrolase,purine nucleoside phosphorylase, and/or purine phosphoribosyltransferase.
 37. The method of claim 36 wherein the protozoan parasiteis Plasmodium.
 38. The method of claim 36 wherein the infection is amalarial infection.
 39. A method for killing parasites comprisingadministering to the parasite an amount of a compound of claim 23effective for inhibiting at least one purine nucleoside hydrolase,purine nucleoside phosphorylase, and/or purine phosphoribosyltransferase.